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A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase. The apoenzyme of D-amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo-D-amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 x 10(-8) M was calculated. The kinetics of formation of the apoprotein-FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two-stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4-6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast D-amino acid oxidase appears to be of the type Apo + FAD in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.

Intracerebral distribution of DL-2-amino-phosphonopentanoic acid (AP5) and the dissociation of different types of learning. Chronic intraventricular infusion of the selective NMDA receptor antagonist AP5 appears to cause an impairment of spatial but not visual discrimination learning. However, Goddard (1986) has questioned whether this dissociation in task-selectivity reflects a difference in the underlying neural mechanisms or differential drug diffusion. Two experiments conducted to address this issue established (a) that chronic intraventricular infusion of AP5, at a dose sufficient to cause a spatial learning impairment, results in a relatively uniform distribution of the drug across the brain, and (b) that chronic bilateral intracortical infusion at sites very close to visual cortex also fails to impair visual discrimination learning. These findings argue against differential diffusion being a major cause of the sensitivity of spatial but not visual discrimination tasks to AP5, and raises the possibility that representational and procedural memory tasks may depend upon distinct cell-biological mechanisms of plasticity.

Systematic presence of GABA-immunoreactivity in the tubero-infundibular and tubero-hypophyseal dopaminergic axonal systems: an ultrastructural immunogold study on several mammals. Immunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plastic-embedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH-immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.

Prevalence of HIV infection in 646 heroin addicts and outcome of HIV-related conditions in the 266 followed up. In a series of 646 heroin addicts anti-HIV was detected in 428 (66.2%) and HBsAg in 53 (8.2%). Forty-eight (90.5%) of the latter had concomitant chronic HDV infection. Markers of past HBV infection were found in 481 (74.4%). The prevalence of anti-HIV was significantly higher in the 534 subjects with HBV markers than in the other 112 without markers (69.8% versus 49.1%, p less than 0.001). Of the 266 anti-HIV positive subjects followed for 3-48 months (median 12), nine progressed from no disease to persistent generalized lymphadenopathy (PGL), 52 from PGL to AIDS-related complex (ARC) or AIDS (30 and 22 cases respectively), and six from ARC to AIDS. Baseline T4 + cell count was significantly lower and reduction during follow-up significantly greater in heroin addicts with disease progression than in those without.

Effects of sodium-nitroprusside and urapidil on gas exchange and ventilation-perfusion relationships in patients with congestive heart failure. Vasodilators usually decrease arterial PO2 in patients with congestive heart failure (CHF) because of alteration in ventilation-perfusion (Va/Q) relationships. The effects of sodium nitroprusside (SNP) and urapidil (U), a new selective alpha 1-receptor antagonist, were investigated in seven patients with CHF. The distribution of ventilation and perfusion was examined using the multiple gas elimination technique. The haemodynamic responses to SNP and U were similar, cardiac index increasing by 25% with SNP and by 31% with U. Despite a similar increase of mixed venous oxygen tension, the arterial PO2 decreased from 11.3 +/- 0.8 to 9.6 +/- 0.6 kPa (p less than 0.01) with SNP but remained unchanged (11.0 +/- 0.9 vs 11.4 +/- 0.8 kPa, NS) with U. SNP and U both increased perfusion to lung units with Va/Q ratios of 0.1 or less with no change in shunt fraction. The fractional perfusion to total low Va/Q ratios (low Va/Q + shunt) was higher with SNP than with U (14.1 +/- 2.6 vs 9.5 +/- 2.3%, p less than 0.01). The results suggest that gas exchange and Va/Q relationships are altered less with U than with SNP in patients with CHF.

Sequence similarity between opioid peptide precursors and DNA-binding proteins. The opioid peptide precursors, preprodynorphin and preproenkephalin show structure similarity with a transcription factor, hunchback and the putative helix-loop-helix DNA-binding proteins, lil-1, tal and twist. Segments with similarity contain the three enkephalin sequences in preprodynorphin and one in preproenkephalin which are present within heptapeptide repeats characteristic of an alpha-helical coiled-coil structure distinctive of an amphipathic helix-loop-helix DNA-binding motif. Hunchback and the opioid prohormones also have cystein-rich regions characteristic of zinc-finger domains in common.

Noninsulin dependent diabetes mellitus and pregnancy in Mexico. We analyzed 215 consecutive patients with diabetes mellitus and pregnancy, 118 (54.83%) with noninsulin dependent diabetes mellitus (NIDDM), 90 (41.86%) with gestational diabetes mellitus (GDM) and 7 (3.26%) with insulin dependent diabetes mellitus (IDDM). NIDDM and GDM patients had no significant difference in age and body mass index. There were no maternal deaths, nor episodes of ketoacidosis. Maternal and neonatal complications occurred with a similar frequency in NIDDM and GDM. We concluded that in our population, diabetes associated with insulin-resistance occurred in over 96% of our pregnant diabetic patients and was associated with an increased prevalence of maternal and neonatal complications. Earlier perinatal care has to be established in NIDDM patients, and obese young women should be screened to detect GDM from early gestation and advised to reduce weight before pregnancy ensues.

Vaginal birth after cesarean section in rural Tanzania. Eighty-seven of 134 women with a history of previous cesarean section in two rural hospitals in Tanzania had a vaginal delivery after a trial of labor. The incidence of scar-rupture was high: in 9 of 134 cases (6.7%). Maternal death, however, did not occur. It is concluded that a trial of labor is justified, and that the risk of scar-rupture should be balanced with the risk of repeat operations.

Prevention of postcesarean infectious morbidity with a single dose of intravenous metronidazole. The efficacy of intravenous metronidazole for the prevention of postcesarean section infectious morbidity was studied in 100 healthy women, randomly given either the drug or a placebo. The metronidazole group received 1.0 g intravenously, immediately after cord clamping. Among the 50 patients who received metronidazole, endometritis developed in 7 (14%) as it did in 15 (30%) of the placebo group (P less than 0.01); wound infection was found in 1 (2%) and 4 (8%), respectively (P less than 0.01). If both infectious complications are compared together, the difference (16% versus 38%) is more significant (P less than 0.001). Metronidazole was well tolerated by the mother and with this type of administration regimen, the fetus is not exposed to the drug. It is concluded that metronidazole, used as here reported, is effective in reducing the frequency of postcesarean section endometritis and wound infection with the consequent clinical and economic impacts.

Maternal and perinatal complications in neurofibromatosis during pregnancy. A total of ten patients with lesions of neurofibromatosis during pregnancy were followed up for pregnancy complications. Seven cases (70%) had hypertensive disorders of pregnancy; four had severe PET (pre-eclamptic toxemia) including one case of eclampsia, one had mild PET and the other two had only mild gestational hypertension. A total of 60% had preterm labor and in none of these did the baby survive; thus perinatal mortality was 600/1000. Mean gestation was 33.0 weeks and mean birthweight was only 1.924 kg. Thus, neurofibromatosis during pregnancy is associated with poor obstetrical outcome and requires greater care.

Safe epidural anesthesia performed during labor by an obstetrician. One thousand seven hundred eighty-seven epidural anesthesias during labor were performed by obstetricians well trained in the technique by the anesthesiology department. The course of labor was accelerated compared to control group. Vacuum extraction rate was 11.9%. Fetal outcome was favorable. Incidence of major complications was 0.16%. We conclude that in countries where an anesthetist is not routinely available at delivery wards, a safe and efficient epidural anaesthesia could be performed by an obstetrician well trained in administration of epidural anesthesia.

Ureteric injuries associated with gynecologic surgery. We conducted a review of eight ureteric injuries associated with major gynecologic surgery in seven patients over an 11-year period. Our low incidence of 0.36% is comparable with other reports. Diagnosis was made either intra-operatively or postoperatively. Immediate ureteric repair is advocated for all injuries discovered intra-operatively. Attention to preventive measures both before and during gynecological operations will reduce the incidence of ureteric injuries.

Extrauterine pregnancy in Mozambique. Epidemiologic, clinical and management information was collected from the case histories of 103 women operated on for an extrauterine pregnancy at the Department of Obstetrics and Gynaecology of the Central Hospital in Maputo (Mozambique) from April 1st, 1987 to March 31st, 1988. We observed a decline in extrauterine pregnancies (9.76%), an association of pelvic schistosomiasis (3.9%), and a high percentage of women with ruptured tubes and subsequent hemoperitoneum (75.7%).

Premenstrual tension: a placebo-controlled efficacy study with spironolactone and medroxyprogesterone acetate. Forty-three healthy women with a characteristic history of premenstrual tension participated in a placebo controlled, crossover study. The effects of spironolactone (Aldactone) and medroxyprogesterone acetate (Gestapuran) on ten symptoms of premenstrual tension were evaluated. Placebo tablets as well as spironolactone and medroxyprogesterone acetate significantly improved a mood index score (which is a generally accepted method to measure premenstrual symptoms). Spironolactone and medroxyprogesterone acetate were however both significantly (P less than 0.05) better than placebo in relieving the symptoms.

Role of microcolposcopy in the diagnostic evaluation of cervical pre-invasive lesions. The present clinical study was undertaken to assess and compare the value of microcolposcopy and endocervical curettage in the diagnostic workup of patients with abnormal cervical lesions. Microcolposcopic findings were well correlated to histology both on directed biopsy and endocervical curettage specimens. Therefore, both microcolposcopy and endocervical curettage are useful procedures to explore the endocervical mucosa. Microcolposcopy should be mainly indicated in those cases where colposcopy has failed to show any lesion, or when colposcopy is unsatisfactory.

Prevention of osteoporosis by medroxyprogesterone acetate in postmenopausal women. The effect of medroxyprogesterone acetate 10 mg BID alone, conjugated estrogens alone or in a combination regimen for the prevention of osteoporosis was determined in 36 postmenopausal women using single photon densitometry. No significant differences in cortical or trabecular bone mass over time were detected in women between the three treatment groups, although a slight increase in bone mass was noted in women with the combined therapy. Medroxyprogesterone acetate appears efficacious in preventing postmenopausal osteoporosis, and may be especially useful in women with contraindications to estrogen replacement therapy.

The variation of endometrial protein PP14 in different parts of the human endometrium. The levels of the endometrial protein PP14 were shown to vary greatly in different sites in the endometrium (coefficient of variation ranged from 55% to 92%, mean = 73%). The variation was found to have no consistent pattern in the seven subjects studied and was unlikely to be the result of problems related to tissue sampling, processing or assay (intra-assay coefficient of variation of the PP14 assay used was less than 10%). Such a large variation in results suggests that the measurement of PP14 concentration in endometrial tissue may be of limited diagnostic value.

Illegal induced abortion--a continuing problem in Nigeria. Illegal induced abortion is still a major contributing factor to maternal morbidity and mortality in Nigeria. It is very common among school girls who are still ignorant of contraception. A good percentage of the abortions are procured by nonmedically qualified personnel. The author has not only advocated the introduction of sex education into the school curriculum as a redeeming measure, but also the provision of contraception in schools and the liberalization of the abortion law.

Percutaneous ultrasound-guided fetal skin biopsy: a new approach. Our preliminary experience with this unreported percutaneous ultrasound guided method using a fine needle system showed 100% success (seven cases) in obtaining fetal skin biopsies. The fetal scalp was chosen for biopsy. This technique is easy, less invasive, simple, has minimum procedural difficulties and complications and thus it may replace fetoscopy for obtaining fetal skin biopsy.

An unusual cause and treatment of decelerations in fetal heart rate. Decelerations of severe degree were detected during the monitoring of fetal heart rate at term. Decelerations continued despite the usual maneuvers in patient's position. Examination by ultrasound revealed that a loop of umbilical cord was held and squeezed in the fetal fist. The oxytocin infusion stimulated strong and frequent fetal movements which caused the fetal grip on umbilical cord to release and FHR decelerations to disappear. Induced labor progressed rapidly and within 5 h a healthy term infant was delivered.

Surviving child from tubal pregnancy. A case report of a 1150 g child surviving from a tubal pregnancy in the 30th week of gestation, delivered by cesarean laparotomy, is described. A review of the literature disclosed nine other cases.

Unicornuate uterus. True unicornuate uterus is a rare anomaly which is often associated with renal tract anomalies, and may predispose to infertility and pregnancy complications. Three additional cases are reported here. One of the women had associated bony and cardiac anomalies, although there was no good evidence that these were part of a syndrome. The diagnosis in each case was made incidentally during the surgical investigation of abdominal pain, which in one case was caused by an ectopic pregnancy, itself a rare occurrence with unicornuate uterus.

Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy. The yeast alpha 2 protein is a regulator of cell type in Saccharomyces cerevisiae. It represses transcription of a set of target genes by binding to an operator located upstream of each of these genes. The alpha 2 protein shares weak sequence similarity with members of the homeo domain family; the homeo domain is a 60-amino-acid segment found in many eukaryotic transcriptional regulators. In this paper we address the question of whether alpha 2 is structurally related to prototypical members of the homeo domain family. We used solution 1H and 15N nuclear magnetic resonance [NMR] spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of alpha 2 that contains the homeo domain homology. We have obtained resonance assignments for the backbone protons and nitrogens of the entire 60-residue region of the putative homeo domain and for most of the remainder of the alpha 2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3JHN-H alpha coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the alpha 2 fragment consisting of residues 11-23, 32-42, and 46-60 (corresponding to residues 138-150, 159-169, and 173-187 of the intact protein). The positions of these three helices correspond extremely well to those of the Drosophila Antennapedia (Antp) and engrailed (en) homeo domains, whose three-dimensional structures have recently been determined by NMR spectroscopy and X-ray crystallography, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effectiveness of neuro-electric therapy in drug resistant endogenous psychoses]. A retrospective chart review of 50 pharmacotherapeutically resistant patients was performed after treatment with NET in 1986-1988. 28 patients suffered from schizophrenia and 22 from affective psychosis. In contrast to literature where NET as therapy of first choice has favourable results in depression in this study 60.7% of the treatment resistant acute schizophrenics responded well to NET. 3 months after discharge from hospital 9 schizophrenics (32.1%) but only 3 patients with affective psychosis (13.6%) presented a 'good' outcome (full remission). A longer duration of schizophrenia (more than 5 years since first manifestation) and a good response to neuroleptics in history was predictive for a good actual NET response (14 of 17 patients), whereas 7 of 11 patients suffering from schizophrenia less than 3 years without any period of full remission on neuroleptics were also non-responders to NET.

Congestive heart failure in patients with normal left ventricular systolic function: a manifestation of diastolic dysfunction. Diastolic dysfunction is a relatively common problem that may be mild and asymptomatic or may present with congestive heart failure and severe disabling symptoms. It is frequently due to coronary artery disease or left ventricular hypertrophy and it is especially common in the older population. The pathophysiology is related to increased left ventricular passive stiffness and impaired or slowed myocardial relaxation. Patients with diastolic dysfunction are best treated with calcium channel blocking agents or beta-blocking agents (drugs that are generally avoided in patients with significant systolic dysfunction). Most treatment is based on symptomatic relief, and therefore periods of cautious trial and error are the rule. Congestive symptoms are treated with agents that reduce pulmonary venous pressure; in general positive inotropic agents and arterial vasodilators are not useful in heart failure that is due to diastolic dysfunction.

Parental origin and mechanism of formation of polysomy X: an XXXXX case and four XXXXY cases determined with RFLPs. The parental origin and mechanism of formation of polysomy X were studied in five cases (one case of 49,XXXXX; four cases of 49,XXXXY), using various X-linked restriction fragment length polymorphisms as genetic markers. Segregation and densitometric analyses on the polymorphic DNA fragments revealed that, in all five cases, the additional X chromosomes are of maternal origin and the mechanism of formation is most probably a result of three non-disjunctions during maternal meiotic divisions: once at the first meiosis and simultaneously twice at the second meiosis. The identical origin and the identical mechanism of formation among the five cases are unlikely to be coincidental and suggest a common cause in the mothers of the five cases.

Polymorphism in a ferritin H gene from chromosome 6p. This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes. A collection of 2,000 lambda phage-carrying human single-copy inserts (greater than 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two human-hamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs; 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100-150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.

DNA polymorphism haplotypes of the human lipoprotein lipase gene: possible association with high density lipoprotein levels. Lipoprotein lipase (LPL) plays a central role in the metabolism of lipoproteins by hydrolyzing the core triglycerides of circulating very low density lipoproteins and chylomicrons. The enzyme is encoded by a gene about 30 kb in size located on the short arm of human chromosome 8. We have determined the locations of the four common DNA polymorphisms along the gene, including a polymorphism that occurred only among an American black population examined. These restriction site polymorphisms were used for haplotype analysis of Mediterranean and US black families. Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium between these sites. No correlation was observed between the level of linkage disequilibrium and the physical distance of the polymorphic sites. The polymorphism information content of the haplotypes ranged from 0.65 to 0.74, thereby constituting a relatively useful genetic marker on chromosome 8. We tested for possible associations between the polymorphisms and circulating lipoprotein phenotypes in a population of 139 Caucasians undergoing coronary arteriography and 50 of their spouses. Some possibly significant associations between LPL gene polymorphisms and levels of high density lipoprotein cholesterol (P = 0.015) and total plasma cholesterol (P = 0.025) were observed. In contrast to a previous report, we found no significant associations with the levels of plasma triglycerides.

Genetic mapping of 12 marker loci in the Xp22.3-p21.2 region. To provide a more precise genetic map of the p22.3-p21.2 region on the short arm of the human X chromosome, we performed multilocus linkage studies in an expanded database including 31 retinoschisis families and 40 normal families. Twelve loci from this region were examined. Although significant lod scores were observed between various pairs of markers by two-point linkage analysis, the confidence limits were found to be broad. The most likely gene order on the basis of multilocus analysis was Xpter-DXS89-DXS85-DXS16-(DXS207,DXS43++ +)-DXS274-(DXS41, DXS92)-ZFX-DXS164-Xcen. All other alternative orders were excluded by odds of at least 40:1.

Rett syndrome: exclusion mapping following the hypothesis of germinal mosaicism for new X-linked mutations. The hypothesis of germinal mosaicism in the unaffected mother of two half-sisters affected with Rett syndrome is postulated to explain the unusual recurrence of this genetic disorder affecting only females (1/10,000); it might be caused by new X-linked mutations with lethality in male fetuses. The analysis of 34 X-linked restriction fragment length polymorphisms (RFLPs) in these two affected females and in their unaffected mother and half-brother, together with the reconstruction of phase for 15 informative RFLPs in somatic cell hybrids retaining a single X chromosome from each female, has made it possible to exclude some regions of the X chromosome as possible sites of the mutation(s) causing Rett syndrome.

A phase II study of taxol in patients with malignant melanoma. Based on results of a phase I study demonstrating antitumor activity of taxol in patients with melanoma, 34 patients with documented metastatic melanoma received taxol, 250 mg/m2, as a 24-hours infusion, repeated every 21 days, in this phase II study. All patients received premedication with dexamethasone, diphenhydramine and cimetidine. Four patients experienced anaphylactic reactions and stopped treatment. Other significant toxicity of this drug included short-lived but severe neutropenia (less than 1,000/mm2) and peripheral neurotoxicity. Four of 28 evaluable patients demonstrated objective response (14%) (confidence interval, 4%-33%) including 3 complete responses and 1 partial response. Two complete responders are continuing at 25+ and 38+ months after achieving CR. Minor evidence of anti-tumor activity was noted in five additional patients. Taxol has significant activity in melanoma and should be further studied in combination with other agents in this disease.

Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene. The product of the proliferating cell nuclear antigen (PCNA) gene is the co-factor of DNA polymerase delta, which is required for cellular and viral DNA replication. Its steady-state mRNA levels are growth-regulated in young human diploid fibroblasts (HDF) as well as in many other cell types. In senescent HDF, PCNA mRNA is not detectable. However, the PCNA gene is transcribed in senescent HDF as efficiently as in young cells. Furthermore, PCNA hnRNA is easily detectable by reverse transcriptase-polymerase chain reaction in both senescent and young HDF, and the levels are essentially similar. These results indicate that in senescent HDF which are incapable of synthesizing cellular DNA, one of the genes coding for a protein of the DNA-synthesizing apparatus is still transcribed, but the product fails to be processed into mature mRNA.

Genetic distinction between sterol-mediated transcriptional and posttranscriptional control of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterols reduce the activity of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) transcriptionally by inhibiting the synthesis of reductase mRNA and posttranscriptionally by accelerating degradation of the enzyme. We and others have described mutant lines of Chinese hamster fibroblasts that are completely resistant to sterol-mediated repression of transcription of HMG-CoA reductase as well as two other sterol-regulated genes, HMG-CoA synthase and the low density lipoprotein (LDL) receptor. In the current studies, we show that one line of sterol-resistant mutant cells (SRD-3 cells) retains the ability to slow the degradation of HMG-CoA reductase by 7-fold in response to treatment with compactin, an inhibitor of reductase that blocks sterol synthesis. The compactin effect is reversed by exogenous sterols. Similar results were obtained with another mutant line of sterol-resistant cells (SRD-2 cells) whose defective transcriptional regulation is attributable to a different gene than that in the SRD-3 cells, as determined by complementation analysis. These data indicate that the gene products that are defective in the SRD-3 and SRD-2 cells are not required for the sterol-mediated regulation of degradation of HMG-CoA reductase. Thus, mammalian cells possess at least two genetically distinct mechanisms, one transcriptional and the other posttranscriptional, for sensing and responding to the intracellular level of sterols.

Assessment of beta-1 selectivity of bisoprolol and atenolol by means of their influence on the lipolytic response to an infusion of terbutaline. We have investigated the beta-1 selectivity of a new beta-blocker, Bisoprolol, by comparing its effect on lipolysis induced by intravenous terbutaline infusion with that of Atenolol. At a dose of 5 mg, Bisoprolol had virtually no beta-2 blocking activity as measured by free fatty acid (FFA) release during terbutaline infusion. At a dose of 10 mg, Bisoprolol had a small but statistically insignificant effect on FFA release similar to 50 mg Atenolol. At a dose of 20 mg, Bisoprolol had significant beta-2 blocking activity. At lower doses, therefore, Bisoprolol is a very selective beta-blocker.

Close linkage of bipolar disorder to chromosome 11 markers is excluded in two large Australian pedigrees. The relationship between bipolar disorder and chromosome 11 markers remains uncertain. Whilst re-analysis of the Amish pedigree weakened previous evidence for close linkage (but could not exclude the possibility of genetic heterogeneity), a recent French study has found a significant association between this condition and tyrosine hydroxylase polymorphisms. We aimed to determine if bipolar disorder in two large Australian pedigrees (of Irish and English extraction respectively) was linked to these markers. Of the 84 family members available for testing, nine were diagnosed as bipolar I, one as bipolar II and six had recurrent unipolar depression. Linkage of bipolar disorder and recurrent depression to the chromosome 11p15 markers c-Harvey ras, insulin and tyrosine hydroxylase was tested using a series of genetic models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective status (ranging from bipolar I alone to all affective illnesses). Close linkage to these markers was strongly excluded using each model and definition. The findings also persisted when a wide range of rates of 'sporadic' (non-genetic) presentations of illness were incorporated in the analysis. These results are consistent with other recent studies indicating that bipolar disorder is not linked to chromosomal region 11p15.

Ontogeny of somatostatin receptors in the rat somatosensory cortex. The distribution and density of SRIF receptors (SRIF-R) were studied during development in the rat somatosensory cortex by in vitro autoradiography with monoiodinated [Tyr0-DTrp8]S14. In 16-day-old fetuses (E16), intense labeling was evident in the intermediate zone of the cortex while low concentrations of SRIF-R were detected in the marginal and ventricular zones. The highest density of SRIF-R was measured in the intermediate zone at E18. At this stage, labeling was also intense in the internal part of the developing cortical plate; in contrast, the concentration of binding sites associated with the marginal and ventricular zones remained relatively low. Profound modifications in the distribution of SRIF-R appeared at birth. In particular, a transient reduction of receptor density occurred in the cortical plate. During the first postnatal week, the density of receptors measured in the intermediate zone decreased gradually; conversely, high levels of SRIF-R were observed in the developing cortical layers (II to VI). At postpartum day 13 (P13), a stage which just precedes completion of cell migration in the parietal cortex, the most intensely labeled regions were layers V-VI and future layers II-III. From P13 to adulthood, the concentrations of SRIF-R decreased in all cortical layers (I to VI) and the pattern of distribution of receptors at P21 was similar to that observed in the adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cetirizine on histamine- and leukotriene D4-induced bronchoconstriction in patients with atopic asthma. Cetirizine, a derivative of hydroxyzine, is a new compound with potent antihistaminic property without antiserotonin and anticholinergic activities. The effect of both a single dose (15 mg) and 7 days of treatment (15 mg twice daily) with cetirizine, a potent H1 antagonist on bronchoconstriction induced by histamine and leukotriene D4 (LTD4) has been examined in 10 patients with mild atopic asthma in a placebo-controlled, double-blind, crossover study. Cetirizine, after a single dose and 7 days of treatment with placebo, the geometric mean values of the provocative concentration of histamine causing a 20% fall in FEV1 (millimolars) were 1.60 (95% confidence interval, 0.82 to 3.11) and 1.67 (0.77 to 3.65), compared with 118.07 (77.22 to 180.54) (p less than 0.0001) and 53.16 (20.50 to 137.84) after cetirizine administration (p less than 0.0002). The mean inhibition after a single dose was twofold higher than after 1 week of treatment (p less than 0.05). After a single dose and 7 days of treatment with placebo, the geometric mean values of the provocative concentration of LTD4 causing a 20% fall in FEV1 (micromolars) were 2.26 (1.74 to 2.94) and 2.37 (1.77 to 3.17), compared with 3.90 (2.60 to 5.86) (p less than 0.05) and 3.21 (2.28 to 4.52) after cetirizine administration. This result suggests that cetirizine is a potent H1 antagonist in the human airways. Diminished activity after 1 week of treatment suggests subsensitivity of H1 receptors developing in human airways. The small protective effect after a single dose against LTD4-induced bronchoconstriction indicates a nonspecific rather than a specific receptor antagonism.

Effect of H1-receptor blockade on late cutaneous reactions to antigen: a double-blind, controlled study. This study is of the effect of the blockade of histamine H1 receptors by a long-acting antihistamine on the immediate and late clinical response to antigen (Ag) and on the recruitment of eosinophils in the late-phase cutaneous reaction. Ten adult volunteers with late-phase reactions to the intradermal injection of either Dermatophagoides pteronyssinus or Phleum pratense (timothy) pollen performed a double-blind, crossover study. Each volunteer took astemizole, 10 mg, or identical placebo, daily for 2 weeks. Ag in the concentration that induced a late reaction in the screening visit was injected intradermally at the end of each drug period. The early reaction was measured serially for 30 minutes and the late reaction at 4 and 6 hours. Biopsies of the Ag and control sites were also performed at 6 hours. After a 6-week washout period, subjects then took the opposite medication for 2 weeks and returned for skin testing and biopsy. Skin testing demonstrated that astemizole inhibited the immediate response to both histamine and allergen but had no effect on the late response at 4 hours and at 6 hours. Biopsy specimens revealed no significant effect on eosinophil recruitment at 6 hours. We conclude that histamine H1-receptor blockade has no effect on the late cutaneous reaction to Ag.

The effect of inhaled ipratropium bromide alone and in combination with oral terfenadine on bronchoconstriction provoked by adenosine 5'-monophosphate and histamine in asthma. The aim of this study was to investigate the effect of terfenadine, an antihistamine, 180 mg orally, the anticholinergic drug, ipratropium bromide (IB), 0.5 mg nebulized aerosol, the combination of these two drugs, and placebo tablets and aerosol on histamine- and adenosine 5'-monophosphate (AMP)-induced bronchoconstriction in a randomized, double-blind fashion. Airway response was evaluated as FEV1. After placebo, the geometric mean (GM) provocative concentration causing a 20% in FEV1 from the postsaline baseline value (PC20) for histamine and AMP was 0.63 and 5 mg/ml, respectively. Terfenadine displaced the FEV1 concentration-response curves obtained with both histamine (GM PC20 values increasing to 26.92 mg/ml) and AMP (GM PC20 values increasing to 26.7 mg/ml) to the right. IB had a small, but significant, protective effect against the fall in FEV1 produced by histamine and AMP, the GM PC20 values increasing to 1.69 and to 12.6 mg/ml, respectively. Terfenadine and IB in combination produced protection against histamine and AMP that was more than the production produced by either drug alone, the GM PC20 values increasing to 54.76 and 47.7 mg/ml, respectively. There was no correlation between degree of bronchodilatation induced by active treatments and concentration ratios for AMP or histamine. These data suggest that histamine release and vagal reflexes both contribute to AMP-induced bronchoconstriction in clinical asthma in man.

Characteristics of CR3-mediated aggregation in human eosinophils: effect of priming by platelet-activating factor. We have used double-color fluorescence-activated cell sorter analysis to characterize the homotypic aggregation response of human eosinophils (EOs). With this method, we demonstrate for the first time that EOs are able to form stable aggregates. The aggregation response induced by the phorbol ester, phorbol myristate acetate (PMA), was low and was not primed by platelet-activating factor (PAF). In contrast, the aggregation response induced by opsonized particles was markedly enhanced after priming with PAF. Additional experiments with several blocking monoclonal antibodies indicate that the CR3 receptor present on human EOs mediates the homotypic aggregation induced by PMA and by small opsonized particles through a putative "cell-adhesion" site on CR3, which binds to its counter structure on the opposing cell. The signal that initiates this binding event is generated after PMA addition or activation of the iC3b-binding site and is not sensitive for priming by PAF. The priming by PAF of the aggregation response induced by opsonized particles is restricted to an action on the iC3b-binding site, possibly by enhancing the affinity for its ligand.

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion. Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits. CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.

Association of various T cell-surface molecules with the cytoskeleton. Effect of cross-linking and activation. The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.

Distinct subsets of accessory cells activate Thy-1+ triple negative (CD3-, CD4-, CD8-) cells and Th-1 delayed-type hypersensitivity effector T cells. The SJL strain of mice possess a unique developmental delay in the ability to exhibit delayed-type hypersensitivity (DTH) responses after immunization with a wide variety of Ag. Similar to other models of DTH, the adoptive transfer of syngeneic Ag-pulsed macrophages from DTH-responsive mice into these DTH-unresponsive mice results in the activation of Ag-specific, CD4+ DTH effector Th1 T cells. The absence of other defects in APC-dependent immune responses indicate that the macrophages is the sole APC required for the induction of DTH effector T cells in SJL mice. The defect occurs during the sensitization phase of the DTH response; however, it has not been determined whether a Th cell, which is required for the induction of CD4+ DTH effector T cells, was present in the DTH unresponsive SJL mice. In this study, we have determined that the Thy-1+ helper cell is induced upon Ag stimulation of nonresponder mice and present evidence for the existence of an accessory cell distinct from the macrophage that induces CD4+ DTH effector T cells. Our data indicate that CD4+ DTH effector T cells are induced in an Ag-specific and MHC-restricted manner by an adherent macrophage that expresses the Mac-1+, Mac-2-, Mac-3+, I-A+ phenotype. Adoptive transfer of as few as 100 of the Mac-1+, Mac-2-, or Mac-3+ subsets from DTH responsive donors to DTH unresponsive recipients is able to overcome the DTH deficit. The activation of CD4+ DTH effector T cells in the SJL mouse cells also requires a Thy-1+, Lyt-1+, CD3-, CD4-, CD8-, helper cell. In contrast to the Mac-1+, Mac-3+, I-A+ accessory cell, this helper cell requires an adherent, irradiation resistant, accessory cell that expresses the Mac-1+, Mac-2-, Mac-3-, I-A- surface phenotype for activation. Further, the interaction between this accessory cell and the Thy-1+ helper cell is neither Ag-specific nor MHC restricted. This is the first demonstration of an accessory cell requirement for the Thy-1+, Lyt-1+, B220-, CD4-, CD8-, CD3- DTH Th cell. These data indicate that the activation of the triple negative helper cells and subsequent activation of the CD4+ effector T cells are regulated by two distinct macrophage subpopulations.

Inhibition of epidermal Langerhans cell function by low dose ultraviolet B radiation. Ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells. Immunosuppressive effects of low levels of ultraviolet B (UVB) radiation on cutaneous immune responses have been attributed to deleterious effects of UVB radiation on epidermal Langerhans cells (LC). To determine how UVB radiation modulates LC function we examined the effect of in vitro UVB exposure on LC accessory cell activity and surface phenotype. Exposure of BALB/c murine epidermal cells to low dose (less than or equal to 200 J/m2) UVB radiation in vitro inhibited their ability to support the mitogenic response of unstimulated, accessory cell-depleted splenic T cells to anti-CD3 mAb. LC accessory cell activity was also inhibited when LC were exposed to UVB radiation in situ, although several-fold higher doses of UVB radiation were required to achieve complete inhibition of LC function. This dose-dependent inhibition was mediated through a direct effect on LC that could not be reversed by IL-1 or IL-6 alone or in combination, or granulocyte-macrophage-CSF. TNF-alpha did not inhibit LC accessory cell function in this assay and anti-TNF-alpha neutralizing antibodies did not reverse the inhibitory effects of UVB radiation. UVB irradiated LC failed to participate in the anti-CD3-dependent clustering that normally occurs between T cells and LC during the proliferative response of murine T cells to anti-CD3 mAb, suggesting that UV radiation may interfere with accessory cell function by preventing intercellular adhesion. Two-color flow cytometric studies revealed low levels of the ICAM-1 on freshly isolated LC and some keratinocytes. ICAM-1 expression on LC increased 15 to 20-fold within the first 24 h in vitro and continued to increase during a 72-h culture period. The integrin LFA-1 was not identified on freshly isolated or cultured LC but was detected on responding T cells. Prior exposure of LC to UVB radiation (50 or 100 J/m2) inhibited the increase in ICAM-1 expression that normally occurs in vitro by up to 70% whereas surface levels of class II MHC Ag, CD45 and Fc-gamma receptors were not affected. Blocking studies revealed that anti-CD3 induced T cell proliferation and T cell-LC cluster formation was inhibited by both anti-LFA-1 and anti-ICAM-1 mAb suggesting that ICAM-1 expressed on LC must bind to LFA-1 on T cells to facilitate proliferative responses of T cells to anti-CD3 mAb. We conclude that the in vitro inhibitory effects of low dose UVB radiation on LC accessory function may result because UVB radiation prevents upregulation of ICAM-1 expression by LC in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro infection with HIV enables human CD4+ T cell clones to induce noncognate contact-dependent polyclonal B cell activation. Eleven (nine CD4+ and two CD8+) protein purified derivative-specific and eight tetanus toxoid-specific T cell clones (TCC), established from the peripheral blood of healthy persons, were cocultured in vitro with irradiated mononuclear cells from patients infected by HIV in the presence of PHA and polybrene. Two weeks post-HIV exposure, all 17 CD4+, but neither of the two CD8+, TCC exhibited integration of HIV in their genoma, as detected by polymerase chain reaction analysis, and released HIV into their supernatants, as detected by measuring both reverse transcriptase activity and p24 Ag. When co-cultured with either autologous or allogeneic B cells, all CD4+ HIV-infected TCC induced the synthesis of extraordinarily high amounts of IgM, IgG, and IgA. In contrast, their noninfected counterparts could provide helper function for Ig synthesis by autologous B cells only in the presence of the specific Ag (or anti-CD3 antibody), and induced allogeneic B cells to synthesize Ig only upon stimulation with anti-CD3 antibody. The supernatants of HIV-infected TCC failed to stimulate Ig synthesis in B cells. More importantly, when HIV-infected clonal T blasts and B cells were cultured in different chambers separated by a millipore membrane, permeable to molecules but not to cells, Ig synthesis did not occur. The Ig synthesis induced by HIV-infected TCC was also markedly inhibited by the addition in culture of either anti-CD4 or anti-LFA-1 antibody. In contrast, HIV-infected TCC maintained their ability to provide helper function for Ig synthesis in the absence of any stimulus, even after fixation with p-formaldehyde. These data demonstrate that in vitro infection with HIV enables human T cells to stimulate Ig synthesis by B cells by an Ag-nonspecific, MHC-unrestricted, contact-dependent mechanism. This may explain, at least in part, the hypergammaglobulinemia and other phenomena related to polyclonal B cell activation frequently seen in HIV-infected persons.

Inflammatory edema induced by interactions between IL-1 and the neuropeptide calcitonin gene-related peptide. The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator with a long duration of action. CGRP is widely distributed and is present in perivascular nerves of tissues that include skin and the synovium. In this study we have investigated the possibility that CGRP can modulate the inflammatory actions of the cytokine IL-1 by using an inflammatory model in rabbit skin. The intradermal injection of IL-1 (1.4 x 10(-14) mol/site) alone stimulated little edema formation. However, when IL-1 was injected with CGRP (10(-11) mol/site), a highly significant edema was observed, and neutrophil accumulation induced by IL-1 was potentiated. These results suggest that the action of IL-1 as a potent mediator of increased microvascular permeability is only observed when skin blood flow is increased in this model. This was confirmed by experiments in which PGE2 (3 x 10(-9) mol/site) at a dose with a similar duration of vasodilator action as CGRP (10(-11) mol/site) also potentiated edema induced by IL-1. Further experiments investigated the mechanism by which IL-1 increased microvascular permeability. Edema induced by IL-1 was dependent on de novo protein synthesis and the presence of circulating neutrophils. However, selective platelet-activating factor and histamine H1 antagonists had no inhibitory effect on this response. Thus it appears that when a microvascular bed is dilated by the long-lasting vasodilator CGRP, edema induced by IL-1 is clearly observed. These results highlight a potentially important synergistic interaction between cytokines and neuropeptides in inflammation.

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18). C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.

Polymorphism of the human Ig VH4 gene family. In this study, we analyze the human VH4 gene family and find it to exhibit a level of polymorphism similar to that of the much larger VH3 family. A cloned VH4 probe detected an average of 10 hybridizing BgIII restriction fragments in genomic DNA derived from 75 unrelated individuals and a total of 15 distinct bands. Of these 15 restriction fragments, 12 were polymorphic, as demonstrated by band absence in some individuals. Oligonucleotide probes specific to CDR1 and CDR2 sequences of known VH4 genes detected limited numbers of bands and revealed sequence polymorphisms that correlated with several of the RFLP detected by the cloned probe. The prevalence of the individual polymorphic restriction fragments was highly variable, ranging from 1% to 97%, with a mean prevalence of 51%. These values resemble those previously observed among VH3 elements. Analysis of linkage disequilibrium suggests that most VH4 gene segments are in genetic equilibrium. These results indicate that the VH4 loci, like those of VH3, are dominated by relatively few, perhaps two to four, alleles/locus and further suggest that the haplotype organization of the human VH locus is very complex.

Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis. Pretreatment of the human melanoma cell line, A375, and the human colon carcinoma cell line, HT-29, with certain cytokines was found to increase the vulnerability of these cells to monocyte-mediated killing. This activity was found to correlate with increased expression of intercellular adhesion molecule-1 (ICAM-1) on the tumor cells and was blocked by anti-ICAM-1 antibodies. Both IFN-gamma and TNF induced large increases in the ICAM-1 expression on both cell lines and increased the susceptibility of the tumor cells to monocyte-mediated killing. IFN-alpha and IL-1 beta, however, induced only small increases in ICAM-1 expression and enhanced the lysis of the A375 cells but not the HT-29 cells by monocytes. These differences may be the result of a higher basal expression of ICAM-1 found on the A375 cells when compared with the HT-29 cells. These data indicate that regulation of ICAM-1 expression on tumor cells can alter the vulnerability of these cells to lysis by monocytes.

AIDS in Edinburgh drug users: observations on the epidemic and implications for its future management. We are now witnessing the anticipated explosion of cases of AIDS resulting from the epidemic of HIV infection among Edinburgh drug users in the first half of the last decade. We expect the number of new cases of AIDS to continue to increase, although the rate at which they do so may be mitigated by intervention which slows the rate of progression to AIDS. There is evidence that current management of patients may postpone a diagnosis of AIDS until later in the natural history of HIV infection when immunity is very low as manifested by CD4+ lymphocyte counts. Health care planners need to provide resources for an increasing number of HIV-infected persons who have not yet fulfilled the definition of AIDS but who nevertheless require extensive resources both in the community and in hospital.

Acute rheumatic fever in human immunodeficiency virus infection. A 25-year-old homosexual man with a childhood history of rheumatic heart disease presented with painful joints, fever and chest pain. He was diagnosed as having acute rheumatic fever and was found to be HIV antibody-positive. His illness responded to conventional treatment but he had a persistently low CD4 lymphocyte count and was started on zidovudine. Interpretation of the significance of a low CD4 lymphocyte count is problematic in a patient with coincident rheumatic fever and HIV infection as both conditions can cause CD4 lymphopenia.

T helper/inducer (CD4+) cells prestimulated with PPD induce monocytes to produce interleukin-1 beta. We obtained peripheral blood mononuclear cells (PBMC) from four healthy, tuberculin purified protein derivative (PPD) reactive donors and cultured these cells in media containing PPD (low dose = 200 ng/ml or high dose = 1 micrograms/ml). Five days after the addition of PPD, T cells were isolated, washed, and added to autologous adherent cell cultures at a 1:1 ratio. Adherent cells were then cultured for 24 h in media only (baseline), media plus lipopolysaccharide (LPS, 2 micrograms/ml; positive control), or media containing the prestimulated T cells. After 24 h, supernatants were harvested and interleukin 1 beta (IL-beta) levels were assayed by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The results show that T cells prestimulated with low dose PPD (200 micrograms/ml) did not induce IL-1 production by adherent cells (mean increase over baseline 0.2 +/- 1.3 standard deviation [SD] ng/ml, P = 0.61). However, T cells prestimulated with high dose PPD (1 microgram/ml) did induce adherent cells to secrete IL-1 beta (mean increase over baseline 1.7 +/- 0.62 [SD] ng/ml, P = 0.01), but this induction was abolished when cell-to-cell contact was prevented by use of double well chambers (mean increase over baseline 0.1 +/- 0.36 [SD] ng/ml, P = 0.69). Prestimulated T helper (CD4+) cells were able to induce monocytes to secrete IL-1 beta but prestimulated CD8+ T cells were not. These data suggest that when T helper (CD4+) cells are sufficiently activated they acquire the ability to induce monocytes to secrete IL-1 beta. Cell-to-cell contact between monocytes and T cells is required. This function of activated T cells may be important in the normal cellular immune response.

Degree of coronary artery disease predicted by exercise testing. The ability of exercise testing to predict the extent of coronary artery disease was examined in 268 male patients undergoing both coronary angiography and bicycle testing with electrocardiography before coronary artery bypass surgery. When maximal ST-depressions limited by symptoms increased from 0 to 4 mm or more, the percentage of patients with 'serious' coronary disease, defined as either triple vessel disease or left main stem stenosis, increased from 50% to 80% (P = 0.0001). The patients in the lowest third of physical work capacity showed only a slightly increased risk of serious disease. This tendency was abolished in patients who were using beta-blockers, whereas the relationship between ST-depression and disease was not affected by this medication. The probability of finding left main stem stenosis in a patient increased from 5 to 30% with increasing ST-depression: beta-blockers did not affect this relationship, but there was no additional predictive effect of implicating the level of physical work capacity. It is concluded that traditional electrocardiography during exercise is of value when selecting patients for angiography, but that the physical work level obtained during the test does not predict the degree of coronary pathology.

Effects of inflammatory cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to endothelial cell monolayers and extracellular matrix proteins. The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood-borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4-8 hr) with recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), or lymphotoxin (LT; also known as TNF-beta), 35-85% of the U937 cells were able to bind. Interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) did not stimulate U937-EC binding, even though IFN-gamma was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937-EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester-treated U937 cells to noncytokine-treated EC. However, that MAb had no effect on U937 cell binding to TNF-alpha-treated EC. Thus U937 cells use both CD11/CD18-dependent and -independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2-20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg-gly-asp (RGD), which is part of one of the cell-binding domains of FN, demonstrated that the RGD-containing peptide almost totally blocked the phorbol ester-induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester-induced binding of U937 cells to EC.

Alterations in cortical [3H]kainate and alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding in a spontaneous canine model of chronic hepatic encephalopathy. Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.

Neural 22-carbon fatty acids in the weanling rat respond rapidly and specifically to a range of dietary linoleic to alpha-linolenic fatty acid ratios. Changing the dietary ratio of the essential fatty acids (EFA), 18:2n6 and 18:3n3, while keeping the amounts of other fatty acids in the diet constant can rapidly and specifically alter the proportions of n6 and n3 22-carbon fatty acids in the brain of the weanling rat. A dietary 18:2n6/18:3n3 ratio of 165 versus 1.8 caused higher n6 and lower n3 22-carbon fatty acid levels, without changing total 22-carbon fatty acid levels, in phosphatidylethanolamine and phosphatidylcholine from several neural membrane fractions. This was apparent after only 2 weeks and showed no sign of plateauing after 12 weeks. Other neural fatty acids were essentially unaffected. The three most abundant 22-carbon fatty acids responded somewhat differently to increments in the dietary 18:2n6/18:3n3 ratio (1.8, 9, 36, and 165). Levels of 22:4n6 increased by similar absolute amounts for each four-fold increase in dietary 18:2n6/18:3n3 ratio; in contrast, the largest absolute changes in 22:5n6 and 22:6n3 levels occurred as the 18:2n6/18:3n3 ratio increased from 36 to 165. This study shows that the 18:2n6/18:3n3 ratio of diets high in fat (40% of energy) and adequate in EFA, both typical of diets in developed countries, can substantially and relatively quickly affect the 22-carbon fatty acids in the brain, even after the rapid accumulation of these fatty acids during neural growth has ceased.

L-phosphoserine, a metabolite elevated in Alzheimer's disease, interacts with specific L-glutamate receptor subtypes. L-Phosphoserine is one of the phosphomonoesters elevated in Alzheimer's disease brain and has close structural similarity to L-glutamate. This study attempts to define precisely the actions of L-phosphoserine at L-glutamate receptor subtypes. L-Phosphoserine is shown to bind to N-methyl-D-aspartate and kainic acid receptor subtypes, but not to the quisqualic acid subtype. Studies of [3H]MK-801 binding in the presence and absence of L-glutamate and glycine show L-phosphoserine to be a competitive N-methyl-D-aspartate antagonist. The IC50 of L-phosphoserine in these studies varies from 373 to 721 microM. This may indicate a physiologically relevant action of L-phosphoserine in Alzheimer's disease brain because whole brain concentrations may reach over 1 mM.

The turnover of rat cortical alpha 1-adrenoceptors is not modified by repeated electroconvulsive treatment. The effect of repeated treatment with electroconvulsive shock (ECS) on the turnover of cortical alpha 1-adrenoceptors in rats was measured using the N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-induced irreversible receptor inactivation method. Repeated treatment with ECS did not affect parameters (the synthesis rate constant r, the degradation rate constant k) of alpha 1-adrenoceptor turnover. Because increase in the density of alpha 1-adrenoceptors in the ECS-treated group disappears later during measurement of turnover, several calculation possibilities were discussed. The present data confirm that repeated treatment with ECS produces a short-lasting up-regulation of cortical alpha 1-adrenoceptors, but does not affect the turnover of this receptor type.

Beta-adrenergic effects on plasma and brain large neutral amino acids are unaltered by chronic administration of antidepressants. Effects of isoproterenol (3 mg kg-1, i.p. for 60 min) and salbutamol (3, 10 mg kg-1, i.p. for 60 min) on large neutral amino acid concentrations in rat plasma and brain were assessed. Phenylalanine, leucine, isoleucine, and valine were measured by gas chromatography with electron-capture detection; tyrosine and tryptophan were measured by HPLC with electrochemical detection. These drugs induced increases in brain tryptophan, tyrosine, phenylalanine, and valine and decreases in plasma tryptophan, tyrosine, leucine, isoleucine, and valine. Effects of salbutamol (3 mg kg-1, i.p. for 60 min) were assessed following chronic administration of phenelzine sulfate and desipramine.HCl (each drug 10 mg kg-1 per day, s.c. via Alzet 2ML4 osmotic minipumps for 28 days). There were no effects of these antidepressants on basal levels of large neutral amino acids in brain and plasma. In both brain and plasma, salbutamol-induced changes in large neutral amino acids were unaffected by these antidepressants. The results indicate that beta-adrenoceptor-regulated availability of plasma and brain large neutral amino acids is unaffected by chronic administration of tricyclic or monoamine oxidase inhibitor antidepressants.

Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.

Pretreatment of cerebellar granule cells with concanavalin A potentiates quisqualate-stimulated phosphoinositide hydrolysis. The hydrolysis of phosphoinositides (PI) elicited in cerebellar granule cell cultures by agonists of metabolotropic glutamate receptors, glutmate and quisqualate, was enhanced when the cells were pretreated with concanavalin A (Con-A). A similar effect was produced by wheat germ agglutinin, but not by several other lectins tested. Con-A produced a dose-dependent effect (EC50 = 3 microM) and increased the efficacy but not the potency of the agonists. In contrast, Con-A failed to enhance PI hydrolysis evoked by N-methyl-D-aspartate, kainate, carbachol, the calcium ionophore A23187, or 50 mM K+. The Con-A stimulatory effect was prevented by simultaneous pretreatment with the agonists of ionotropic quisqualate receptors quisqualate, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, but not by the antagonist 6-cyano-7-nitroquioxaline-2,3-dione (CNQX). CNQX, which did not inhibit quisqualate-stimulated PI hydrolysis in untreated cells, abolished the component of quisqualate response enhanced by Con-A pretreatment. The pretreatment with Con-A also increased the influx of 45Ca2+ in granule cells stimulated by quisqualate. This increase was inhibited by CNQX. Moreover, the potentiation of PI hydrolysis by Con-A, but not the response to quisqualate alone, was abolished in the absence of Ca2+ and Na+. Pretreatment of granule cells with pertussis toxin inhibited PI hydrolysis stimulated by the metabolotropic quisqualate receptor and the Con-A-potentiated response by the same percentage, but Ca2+ influx induced by quisqualate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Four forms of tyrosine hydroxylase are present in human adrenal medulla. Alternative splicing can result in up to four forms of human tyrosine hydroxylase (HTH) mRNA (mRNAHTH). In the adrenal gland, a major site of catecholamine biosynthesis, the presence of all four mRNAHTH forms is controversial. In the present study, postmortem human adrenal medullary tissue was analyzed for the presence of multiple forms of HTH protein by blot immunolabeling. Electrophoretic transfers were developed with affinity-purified rabbit anti-HTH antibodies raised against peptides that reproduced the unique amino acid sequences predicted by the four mRNAHTH forms. All four of the predicted HTH protein forms were present in the adrenal glands from a diverse sample population.

Molecular biological approaches to the study of heritable osteoarthritis. Certain forms of osteoarthritis (OA) are inherited in a dominant Mendelian pattern suggesting that defects in one or more of the genes encoding for the structural components of articular cartilage may cause premature cartilage degeneration. Recent advances in molecular biology make it possible to examine the hypothesis that inherited OA is caused by mutations in the genes expressed in cartilage. These methods permit the identification of precise molecular defects and, eventually, may provide tests for the definitive diagnosis of inherited OA. Application of these tests to a general population of patients with OA may establish whether common forms of OA are also caused by mutations in these genes.

[The absence of monocyte-granulocyte antigen expression in a case of AML]. The brief record of a 25 y. o. male patient with AML (FAB M1) is shown, in whom the blast cells did not express any of the 5 myeloid antigens or the other-lineage related antigens, as detected with the monoclonal antibodies. The blast cells were induced to express CD13 antigen after a short-term culture in vitro. This result suggests that CD13 antigen can be expressed virtually by all AML cells, since CD13 antigen is known to cover fresh AML cells at the highest incidence. No unusual clinical feature was noted in this patient as AML case. The collected documentation of antigen-free AML cases seems necessary for the relevant understanding of AML heterogeneity.

[Alterations of Lewis related sugar antigens in gastric atypical epithelial lesions (adenomas or dysplasia) with relation to their malignant changes--an evaluation by follow-up cases]. Alterations of carbohydrate chain antigens were investigated immunohistochemical in relation to histological malignant changes on 62 cases of gastric atypical epithelial lesions (adenomas) which were diagnosed as Group-III at the first biopsy and then followed up more than one year. Among 7 carbohydrate chain antigens which are related to Lewis antigens, sialyl Lex-i antigen showed the most impressive findings; The positivity percentage of the first biopsy specimens of Group-III was 6%, however, it raised to 33% in the final biopsy specimens of Group-IV, 50% in the resected specimens of border-line lesions, and 67% in the resected specimens of carcinomas. The results indicate that there exists a close correlation between malignant change of gastric atypical epithelial lesions and alteration of carbohydrate chain in terms of sialylation.

Organ-specific cardiac antibodies: serological markers for systemic hypertension in autoimmune polyendocrinopathy. Circulating organ-specific autoantibodies are serological markers of destruction or impairment of the relevant endocrine tissue cells and may be associated with abnormal hormone levels with or without clinical evidence of overt disease. We sought organ-specific cardiac antibodies in patients with autoimmune polyendocrinopathy because of increasing evidence that the heart has endocrine characteristics (secretion of atrial natriuretic peptide [ANP] and other peptide hormones). Serum samples from 166 patients with polyendocrinopathy, 80 with autoimmunity confined to one gland, and 200 healthy blood donors were tested for these antibodies by means of immunofluorescence on human heart. Skeletal muscle was used to identify cross-reacting antibodies. Organ-specific cardiac antibodies were detected in significantly more of the patients with autoimmune polyendocrinopathy (28 [17%]) than of those with autoimmunity confined to one gland (1 [1%]) or of normal subjects (7 [3.5%]; p = 0.0001). Among the patients with autoimmune polyendocrinopathy, the prevalence of systemic hypertension was higher in those with cardiac autoantibodies than in those without (5/28 [18%] vs 2/80 [3%]; p = 0.01); the same was true for a family history of hypertension (11 [42%] vs 5 [7%]; p = 0.0001). There were no significant differences in mean basal or stimulated ANP concentrations between patients with or without antibodies or between patients and controls. 5 of the 22 antibody-positive patients had ANP concentrations outside the normal range, but these disturbances were not associated with systemic hypertension or a family history of the disorder. Patients with autoimmune polyendocrinopathy can have organ-specific cardiac antibodies, which may represent novel serological markers for an autoimmune form of systemic hypertension in the absence of overt cardiac disease.

Diagnosis of genetic disease by primer-specified restriction map modification, with application to cystic fibrosis and retinitis pigmentosa. Detection of small alterations or abnormalities in genomic DNA (eg, point mutations or small deletions) has become increasingly important in the diagnosis of genetic disease and polymorphism. When a mutation or polymorphism creates a new restriction endonuclease site, it can easily be identified by polymerase chain reaction (PCR) amplification of the DNA region of interest, followed by digestion with the restriction endonuclease. However, useful restriction sites are the exception, and a variety of specialised techniques have been developed to identify subtle DNA abnormalities. We have shown that where a DNA mutation does not create a useful novel restriction site, such a site can be introduced by PCR and specially chosen primers. The approach is simple and inexpensive and should be broadly applicable in the diagnosis of genetic polymorphism and mutation. The technique is illustrated here by the three base-pair deletion responsible for most cases of cystic fibrosis and by detection of the point mutation in the rhodopsin gene that has been associated with some cases of autosomal dominant retinitis pigmentosa.

Neuronal loss in the frontal cortex in HIV infection. In an attempt to elucidate the cause and mechanism of the dementia and other neurological disorders that can occur in HIV-1 infection, we have quantitatively assessed neuronal populations, by means of a stereological technique (the disector), in the frontal cortex of patients with HIV infection. Eleven of sixty-five brains in the Medical Research Council Central AIDS Brain Bank were selected for study. The selected patients died without opportunistic infection or neoplasm affecting the brain; they had HIV encephalitis or minimal changes. We compared their neuronal counts with those of eight control subjects (seven died of systemic illness, one of pontine haemorrhage which did not affect the cerebral hemispheres). The neuronal numerical density was significantly lower in the HIV group than in the control group (mean [SD] 307 [46] vs 499 [113] x 10(2) per mm3; p less than 0.001). This difference represents a loss of about 38%. There was no significant difference between the HIV subgroups, which suggests that neuronal loss occurs in cases of minor pathology as well as in HIV encephalitis. This finding contributes to the understanding of dementia in AIDS patients and has important implications for their future treatment.

Delayed sleep phase syndrome response to melatonin. The actions of melatonin on the sleep-wake cycle were investigated by means of a randomised, double-blind, placebo-controlled trial in 8 subjects with a delayed sleep phase syndrome attending a sleep disorders clinic. In randomised order the subjects received placebo or melatonin 5 mg daily for 4 weeks with a 1 week washout period between the treatments. Drug or placebo was given at 2200 h, 5 h before the mean time of sleep onset determined by pretrial sleep logs. In all 8 subjects sleep onset time (mean advance 82 [range 19-124] min; p less than 0.01) and wake time (117 [10-187] min; p less than 0.01) were significantly earlier during melatonin treatment than during placebo. Mean total sleep time was slightly less on melatonin (8 h 12 min) than on placebo (8 h 46 min). Alertness acrophase calculated from the subjects' ratings of alertness made every 2 h while awake was unaltered. Melatonin may act as a phase-setter for sleep-wake cycles in subjects with a delayed sleep phase syndrome.

Therapeutic effects of genetically engineered toxin (DAB486IL-2) in patient with chronic lymphocytic leukaemia. In DAB486IL-2 the receptor-binding domain of native diphtheria toxin is replaced by human IL-2 sequences. This recombinant fusion protein is selectively cytotoxic for cells bearing high-affinity IL-2 receptors--eg, leukaemic cells. A patient with chronic lymphocytic leukaemia who did not respond to gamma interferon and conventional antileukaemic drugs has responded to DAB486IL-2.

Survival of classic cholera in Bangladesh. During the present cholera pandemic the El Tor biotype of Vibrio cholerae has completely displaced the classic biotype, except in Bangladesh. We studied the distribution of these two biotypes in twenty-four rural districts during epidemics in 1988-89; there was clustering of the classic biotype in the southern region and of the El Tor biotype in all other regions. These findings suggest that the southern coastal region is now (and may always have been) the habitat of classic cholera. The selective distribution of V cholerae O1 biotypes in Bangladesh may have been affected by ecological changes occurring in the country.

Gallstone formation after major abdominal surgery. 84 patients underwent multiple abdominal ultrasound examinations over a median of 36 months (range 6-140). 11 had gallstones at their first ultrasound examination, and were excluded from further analysis. Of the remaining 73 patients, 12 of 47 who underwent major abdominal surgery had gallstones within 14-36 months of operation, compared with 0 of 26 who did not undergo such surgery. The cumulative prevalence of new gallstones within 3 years of major surgery was 28%; no new gallstones were seen from 36 to up to 140 months postoperatively. By univariate and logistic regression analysis, age and major abdominal surgery were the only significant clinical determinants for the appearance of gallstones during follow-up. The findings of this retrospective study indicate that major abdominal surgery may accelerate the development of gallstones in some patients. If confirmed in a prospective study, it may be possible to define groups at high risk of gallstones after surgery and to institute prophylactic measures.

Treatment of systemic vasculitis with pooled intravenous immunoglobulin. The therapeutic effect of a course of high-dose, pooled, intravenous immunoglobulin (IVIg) on disease activity and circulating antineutrophil cytoplasm antibodies (ANCA) was investigated in 7 patients with systemic vasculitis. 5 had active disease despite conventional immunosuppression, and 2 had not received any treatment. All 7 had clinical improvement, which was sustained in 6 and transient in 1. The fall in ANCA concentrations to a mean of 51% of the pre-treatment values was maintained during follow-up. C-reactive protein concentration also dropped considerably. IVIg seemed to confer a useful therapeutic effect without adverse reaction.

Mefloquine-resistant falciparum malaria on the Thai-Burmese border. Mefloquine is the treatment of choice for uncomplicated multiresistant falciparum malaria, and in combination with sulphadoxine and pyrimethamine (MSP) at a single dose of 15/30/1.5 mg/kg, respectively, has been used in Thailand for the past 6 years. In 1985-86, MSP cured over 98% of 5192 patients with falciparum malaria on the Thai-Burmese border. 4 years later we studied the efficacy of MSP in 395 patients at the same location. The cure rate at 28 days was 70.8% (95% Cl 67-77.2%). The proportion of early treatment failures (in whom parasitaemia did not clear) had risen from 0.27 to 3.7% (p less than 0.0001). Failure rates were 50% in children under 6 years old, 29% in the 6-15 age group, and 19% in adults (p less than 0.001). Patients with early treatment failure were retreated with 25 mg/kg mefloquine, but 27% had a further recrudescence of infection within 28 days. The mean (95% Cl) serum mefloquine concentration at the time of first recrudescence was 638 (546-730) ng/ml, a value previously associated with successful treatment. Mefloquine concentrations were no lower in those with recrudescent infections than in age-matched successfully treated patients, suggesting that pharmacokinetic factors were not responsible for the high treatment-failure rate. Plasmodium falciparum has developed resistance to mefloquine rapidly, despite the addition of sulphadoxine and pyrimethamine and strict control of drug administration. The MSP combination should now be abandoned.

MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70-99%) or with mild (0-29%) carotid stenosis. European Carotid Surgery Trialists' Collaborative Group. The European Carotid Surgery Trial is a multicentre trial of carotid endarterectomy for patients who, after a carotid territory non-disabling ischaemic stroke, transient ischaemic attack, or retinal infarct, are found to have a stenotic lesion in the relevant (ipsilateral) carotid artery. Over the past 10 years 2518 patients have been randomised, and the mean follow-up is now almost 3 years among the 2200 thus far available for analysis of the incidence of strokes that lasted more than 7 days. For the patients with "moderate" (30-69%) stenosis on their prerandomisation angiogram the balance of surgical risk and eventual benefit remains uncertain, and full recruitment continues. For 374 patients with only "mild" (0-29%) stenosis there was little 3-year risk of ipsilateral ischaemic stroke, even in the absence of surgery, so any 3-year benefits of surgery were small, and were outweighed by its early risks. For 778 patients with "severe" (70-99%) stenosis, however, the risks of surgery were significantly outweighed by the later benefits: although 7.5% had a stroke (or died) within 30 days of surgery, during the next 3 years the risks of ipsilateral ischaemic stroke were (by life-table analysis) an extra 2.8% for surgery-allocated and 16.8% for control patients (a sixfold reduction, p less than 0.0001). There was also a small reduction in other strokes, and at 3 years the total risk of surgical death, surgical stroke, ipsilateral ischaemic stroke, or any other stroke was 12.3% for surgery and 21.9% for control (difference 9.6% SD 3.3, 2p less than 0.01). The main concern was to avoid disabling or fatal events, and, among severe stenosis patients, 3.7% had a disabling stroke (or died) within 30 days of surgery, an extra 1.1% surgery versus 8.4% control (p less than 0.0001) had a disabling or fatal ipsilateral ischaemic stroke by 3 years, and the total 3-year risk of any disabling or fatal stroke (or surgical death) was 6.0% surgery versus 11.0% control (overall difference 5.0% SD 2.3, 2p less than 0.05); but, for disabling or fatal stroke the control risks seemed to diminish after the first year, so delay of surgery by just a few months after clinical presentation might make this overall difference non-significant.

Prospective cohort study of prone sleeping position and sudden infant death syndrome. Studies of the link between prone sleeping position and sudden infant death syndrome have been criticised on grounds of recall bias and for not taking into account possible confounding effects. To avoid recall bias and to allow measurement of important biological factors a prospective cohort study of the cause of sudden infant death syndrome (SIDS) is being conducted. The infants included are those at high risk of the syndrome as assessed by a perinatal score. Of the 3110 members of the cohort born between January, 1988, and end of March, 1990, 23 infants later died of SIDS. Sleep position information was available for 15 of these. Matched analysis to control for the confounding effects of infant birthweight and maternal age indicated that prone sleeping position was associated with an increased risk of SIDS (OR 4.47 95% Cl [1.30-15.43]). The findings are strengthened by the results of a concurrent retrospective case-control study of 42 SIDS cases in which the prone position was also associated with an increased risk of SIDS (unadjusted OR 3.45 [1.59-7.49]).

Schizophrenia after prenatal exposure to 1957 A2 influenza epidemic. The birth dates of schizophrenic inpatients in eight health regions in England and Wales were reviewed for any effect of the 1957 A2 influenza epidemic. 5 months after the peak infection prevalence, the number of births of individuals who later developed schizophrenia was 88% higher than the average number of such births in the corresponding periods of the 2 previous and the next 2 years. This finding is in accordance with a study from Helsinki and with clinical and neuropathological evidence of aberrant fetal brain development in the pathogenesis of schizophrenia.

Regional cerebral blood flow and cognitive function in patients with chronic liver disease. Subtle impairments of cognitive function may be an important cause of occupational and psychosocial morbidity in patients with chronic liver disease. Correlation of structural brain abnormalities with cognitive deficits has yielded inconsistent results. 10 patients with cirrhotic liver disease were compared with 10 age, education, and intelligence matched control subjects. Neuropsychological assessment revealed significant overall cognitive impairments in cirrhotic patients compared with controls (p = 0.02). Regional cerebral blood flow was measured by single photon emission computed tomography (SPET or SPECT) and showed increased uptake of radiotracer in the right and left posterior parts of the basal ganglia and right occipital lobe, together with reduced uptake in the right anterior cingulate region. The degree of cognitive impairment was directly correlated with functional abnormalities in the basal ganglia and limbic cortex (p less than 0.05). Our results suggest that impaired cognitive status may be associated with abnormalities of regional brain function in patients with chronic liver disease. Since these deficits are clinically inapparent, our findings have important implications for identification and management of patients with chronic liver disease.

New autonomic and sensory neuropathy with loss of adrenergic sympathetic function and sensory neuropeptides. A 30-year-old woman with longstanding dizziness was found to have a severe postural fall in blood pressure and a reduced skin axon-reflex flare response. Autonomic tests indicated selective impairment of adrenergic sympathetic function. Plasma noradrenaline, adrenaline, dopamine, and dopamine beta hydroxylase were undetectable. Skin biopsy specimens showed loss of tyrosine hydroxylase and neuropeptide Y (markers of adrenergic sympathetic fibres) and of substance P and calcitonin gene-related peptide (sensory neuropeptides). A sural nerve biopsy specimen showed severe depletion of unmyelinated fibres. The constellation of losses were compatible with nerve growth factor (NGF) deprivation, which was confirmed on assay. This new syndrome may be explained by loss of trophic action of NGF.

Timing of surgery during menstrual cycle and survival of premenopausal women with operable breast cancer. Timing of operation in relation to menstrual phase might affect outlook in premenopausal women with operable breast cancer. We examined the records of 249 such women treated between 1975 and 1985, and compared overall and recurrence-free survival in those whose operation was 3-12 days after their last menstrual period (LMP) (group 1, n = 75) with those in whom it was 0-2 or 13-32 days after LMP (group 2, n = 174). Overall and recurrence-free survival were greatly reduced in group 1 women (p less than 0.001 for both). Actuarial survival at 10 years was 54% in group 1 versus 84% in group 2. This effect was independent of other factors, was of much the same importance as nodal status in multivariate analysis, was largely confined to histologically node-positive cases, seemed to be greater in women with small tumours (less than or equal to 2 cm), and was seen in patients with oestrogen-receptor positive and negative tumours. Thus phase of menstrual cycle at operation is of great importance for long-term outlook in premenopausal women with breast cancer.

Adaptation of pulse oximetry for fetal monitoring during labour. Application of pulse oximetry to intrapartum monitoring was investigated in 105 women. No adequate reading could be obtained in 44 cases. Two major sources of artifact, related to probe apposition and signal processing, were identified and excluded. The average arterial oxygen saturation from the fetal scalp was 82% (SD 6%), which is higher than has been inferred from pO2 levels. Readings below 60% or the development of an unstable baseline suggest the presence of substantial hypoxia and acidosis.

Who will operate on Africa's 3 million curably blind people? About half the 6 million blind people in sub-Saharan Africa have surgically curable cataract. The available manpower and resources can only provide services for less than 10% of the new blind cataract patients each year, and little is being done for the estimated 3 million "cataract backlog". A serious limiting factor to the development of prevention of blindness programmes is lack of trained manpower. Despite an increase in the number of ophthalmologists trained in cataract surgery (which varies greatly from country to country), this number is not keeping pace with increased demand for eye-care services, especially in large rural populations. Initiatives that will help to overcome this dilemma are specific post-graduate courses in community ophthalmology in Africa, plans to develop a one-year diploma in ophthalmology course for English-speaking West African countries, and a proposal to upgrade a similar course in Zimbabwe. Additionally there is a need for the training of more ophthalmic assistants, cataract surgeons, and nurses in the diagnosis and management of common ophthalmic disorders. Experienced expatriate ophthalmologists also have an important role in the teaching of doctors and ophthalmic assistants how to select patients and carry out successful inexpensive cataract surgery with appropriate technology and limited facilities.

Beta-adrenergic-antagonist drugs in the prevention of gastrointestinal bleeding in patients with cirrhosis and esophageal varices. An analysis of data and prognostic factors in 589 patients from four randomized clinical trials. Franco-Italian Multicenter Study Group. The value of beta-adrenergic-antagonist drug therapy for the prevention of initial episodes of gastrointestinal bleeding in patients with cirrhosis and esophageal varices is uncertain, both positive and negative study results having been reported. In this study, we analyzed data on individual patients from four randomized, controlled trials to assess the efficacy of this treatment. Of the 589 patients studied, 286 received a beta-adrenergic-antagonist drug (propranolol in 203 and nadolol in 83) and 303 received placebo. After two years, the mean (+/- SE) percentage of patients who had had no upper gastrointestinal bleeding was 78 +/- 3 percent in the beta-adrenergic-antagonist treatment group and 65 +/- 3 percent in the control group (P = 0.002). The percentage of patients without fatal bleeding was 90 +/- 2 percent in the treatment group and 82 +/- 3 percent in the control group (P = 0.01). The percentage of patients surviving after two years was 71 +/- 3 percent in the treatment group and 68 +/- 3 percent in the control group (P = 0.34). After age and severity of cirrhosis were taken into account, the survival rate was better in the treatment group (P = 0.09). The percentage of surviving patients who had had no bleeding after two years was 62 +/- 3 percent in the treatment group and 53 +/- 3 percent in the control group (P = 0.04). Both propranolol and nadolol prevented a first episode of bleeding. Severe cirrhosis and especially the presence of ascites were associated with bleeding (P less than 0.001) and death (P less than 0.001) in both groups. The efficacy of beta-adrenergic-antagonist therapy in the prevention of bleeding (P less than 0.001) and of fatal bleeding (P = 0.004) and in the prevention of bleeding or death (P = 0.005) was the same after adjustment for cause and severity of cirrhosis, ascites, and size of varices. Propranolol and nadolol are effective in preventing first bleeding and reducing the mortality rate associated with gastrointestinal bleeding in patients with cirrhosis, regardless of severity.

A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. UDCA-PBC Study Group. In primary biliary cirrhosis the hepatic lesions may result, at least in part, from the intracellular accumulation of potentially toxic endogenous bile acids. Preliminary work suggests that the administration of ursodiol (also called ursodeoxycholic acid), a hydrophilic bile acid without hepatotoxicity, leads to improvement in the condition of patients with primary biliary cirrhosis. We conducted a two-year, multicenter, double-blind trial to compare the efficacy of ursodiol with that of placebo. Patients with biopsy-proved primary biliary cirrhosis were randomly assigned to receive either ursodiol (13 to 15 mg per kilogram of body weight per day) (n = 73) or placebo (n = 73). Treatment failure was defined as a doubling of bilirubin levels to more than 70 mumol per liter or the occurrence of a severe complication (ascites or variceal bleeding) or an adverse reaction. Treatment failed in 6 patients in the ursodiol group, as compared with 13 in the placebo group (P less than 0.01 by Cox regression model). A single patient in each group withdrew because of minor adverse effects. After two years of treatment, the proportion of patients with clinically overt disease decreased only in the ursodiol group (P less than 0.02). The patients treated with ursodiol had significant improvements in serum levels of bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, cholesterol, and IgM (all P less than 0.001); the antimitochondrial-antibody titer (P less than 0.01); and the Mayo risk score (P less than 0.001). Follow-up analysis of 95 liver-biopsy specimens showed a significant improvement in the mean histologic score (P less than 0.002) and in all the characteristic histologic features except fibrosis only in the group given ursodiol. Ursodiol is a safe and effective treatment for primary biliary cirrhosis.

Regional distribution of somatostatin binding sites in the human hypothalamus: a quantitative autoradiographic study. Using in vitro quantitative autoradiography and [125I]Tyr0-D-Trp8SRIF 14 as radioligand, we characterized the detailed distribution of somatostatin binding sites in human hypothalamus of both infants and adults. Guanosine triphosphate pretreatment, before incubation, allowed us to detect higher [125I]Tyr0-D-Trp8SRIF 14 binding site densities in hypothalamic structures such as preoptic and anterior hypothalamic areas and ventromedial and dorsomedial nuclei. In contrast, guanosine triphosphate was without effect in the other hypothalamic regions. The regional effects of guanosine triphosphate pretreatment were not different in infant and adult hypothalamus. Scatchard analysis showed that in a guanosine triphosphate-sensitive region (preoptic area) and a guanosine triphosphate-insensitive area (infundibular nucleus), [125I]Tyr0-D-Trp8SRIF 14 bound to a single class of binding sites. Affinities were similar in both regions, not modified by guanosine triphosphate pretreatment and not different in the adult (1.5 +/- 1.2 nM vs 3.2 +/- 2.1 nM for preoptic area and infundibular nucleus, respectively) and infant (0.9 +/- 0.5 nM vs 2.4 +/- 1.7 nM for preoptic area and infundibular nucleus). [125I]Tyr0-D-Trp8SRIF 14 binding sites were widely distributed in the anterior, mediobasal and posterior hypothalamus. Somatostatin 28 was twice as potent as somatostatin 14 to displace [125I]Tyr0-D-Trp8SRIF 14 binding in the preoptic area and infundibular nucleus. However, IC50s were 30 times lower in the preoptic area as compared with the infundibular nucleus. In adult as well as in infant, high densities were found mainly in the diagonal band of Broca, preoptic area and infundibular nucleus. Intermediate densities were localized in the anterior hypothalamic area, ventromedial, dorsomedial and lateral mammillary nuclei. The dorsal hypothalamic area, the paraventricular and medial mammillary nuclei displayed low but measurable densities. The only marked difference in the distribution of [125I]Tyr0-D-Trp8SRIF 14 binding sites in adult vs infant was observed in the medial and tuberal nuclei where the concentrations were seven-fold higher in adult hypothalamus.

Regional and laminar distributions of alpha 1-adrenoceptors and their subtypes in human and rat hippocampus. The distributions of the alpha 1-adrenoceptor and its subtypes (alpha 1A and alpha 1B) in human and rat hippocampus are analysed by quantitative receptor autoradiography. alpha 1-Adrenoceptors are labelled by [3H]prazosin. The alpha 1A subtype is visualized by [3H]prazosin after irreversible blockade of alpha 1B adrenoceptors with chloroethylclonidine or directly by [3H]5-methyl-urapidil. The alpha 1B subtype is investigated by [3H]prazosin binding in the presence of the alpha 1A antagonist 5-methyl-urapidil. Considerable differences in the regional and laminar patterns of alpha 1-adrenoceptors are found between rat and human hippocampi. The rat hippocampus is characterized by a low overall density and a rather homogeneous regional and laminar distribution. This is in contrast to the human pattern, which shows a much higher overall level of alpha 1 receptor density and a restriction of alpha 1 receptors to the CA3 region of Ammon's horn and the dentate gyrus. Moreover, alpha 1A and alpha 1B receptors of the human hippocampus are differentially distributed with the alpha 1A subtype concentrated in the hilus and lucidum layer of CA3, and the alpha 1B subtype concentrated in the molecular layer of the dentate gyrus. Additionally, the distribution of alpha 1 receptors is compared with the distribution of 5-hydroxytryptamine 1A receptors. The subtype specific pattern is correlated with the distribution of glutamatergic systems in the human (but not in the rat) hippocampus. alpha 1A Receptor localization coincides with the target area of the mossy fibre system, and alpha 1B receptors are preferentially localized in the target area of the hippocampal associational fibres and partly of the perforant pathway. This result points to possible interactions between noradrenaline- and glutamate-mediated neurotransmission differentiated by topographically segregated alpha 1-adrenoceptor subtypes.

Ageing changes in the transplants of fetal substantia nigra grafted to striatum of adult rat. Fetal nigral neurons from 16 and 17 gestational days were transplanted into the intact striatum of adult rat. On different post-transplantation days (30-360 days), the structural and immunohistochemical details of the transplants were studied. The grafted neurons matured and showed phenotypical characteristics comparable to that of normal nigral neurons in adult rats until 180 days. Tyrosine hydroxylase-positive neurons were seen not only in the transplant but also in the adjacent host striatum. Tyrosine hydroxylase-positive fibres were also seen extending for a short distance into the host striatum. A large number of synapses in the transplants were of asymmetric type, containing clear round vesicles. These synapses resembled the few intrinsic type present in the normal substantia nigra. On the other hand, the predominant type 2 synapses with pleomorphic vesicles in the normal nigra were infrequently encountered in the transplants. On the 300th day, the cytoplasm of a few of the neurons showed ageing changes in the form of clear spaces, paucity of organelles especially rough endoplasmic reticulum, membrane-bound vacuoles and increase in the lipofuscin population. In addition, localized thickening of the soma and the dendrites were seen in relation to randomly distributed neurons. By 360 days, more than one quarter (26%) of the total neurons showed these changes indicating ageing. The number per unit volume of normal neurons decreased significantly when compared to the transplants on 60 and 90 days. In the substantia nigra of age-matched control, except for an increase in the lysosomal population, other ageing changes were not detectable. The neurons of intact substantia nigra of the host rat, chronologically 4-8 months older than the transplanted neurons, also appeared normal but for lipofuscin granules. The present study provides morphological evidence for rapid ageing of neurons in the long term nigral transplants. These observations raise fresh doubts regarding permanent survival of grafted neurons in the host brain. Studies so far conducted are after prior nigral lesions. Trophic factors following lesions of the host tissue may have influenced the long term survival of the transplanted neurons. On the other hand, such changes may have been missed since no detailed morphological investigations of the long term transplants have been done so far.

Anatomical and behavioral comparison of unilateral dopamine-rich grafts implanted into the striatum of neonatal and adult rats. The anatomical and functional characteristics of dopamine neuron-rich grafts implanted into rat pups were compared with those of identical grafts implanted into adult hosts. The host nigrostriatal dopaminergic pathway was unilaterally destroyed by an intrahypothalamic injection of 6-hydroxydopamine. This was followed five days later by the implantation of a cellular suspension obtained from rat embryonic mesencephali. Identical operations were carried out on adult and infant (PD3) rats. The survival rate of implanted tyrosine hydroxylase-positive cells was lower in the neonates. On the other hand, in the neonate hosts, surviving immunoreactive cells migrated extensively throughout the host striatum coursing preferentially below the corpus callosum and towards the subependymal zone. The structural integrity of the host parenchyma was well maintained after the neonatal implantation, in contrast to that observed in the adults. Despite a difference in the cell survival rate, there was no major difference in reinnervation density between the two types of host. The functional capacities of the implants were evaluated by measuring the rotational responses of the animals to dopaminergic agonists. The implants compensated the lesion-induced contralateral rotational response to the mixed agonist apomorphine and the D1 agonist SCH-38393 in both neonates and adults. However, the response to the D2 agonist LY-171555 was not significantly attenuated by the implant. The ipsilateral rotational response to amphetamine observed in the lesioned animals was also compensated and even reversed by the graft. It is concluded that with respect to rotational behavior, similar functional benefits were observed following adult stage or neonatal implantation, despite differences in their anatomical development.

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland. The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.

Non-uniform responses to Ca2+ along the frog neuromuscular junction: effects on the probability of spontaneous and evoked transmitter release. Spontaneous and evoked transmitter release activity was studied during selective application of Ca2+ in proximal (near the first contact of the axon on the muscle fiber) and distal regions of the frog neuromuscular junction. A new technique called "Microperfusion" was developed, which allowed us to apply a 30-microns-wide Ca2+ stream from an external pipette. The spread of this Ca2+ stream was monitored by adding Blue Dextran (40 mg/ml) to the Ca2+ solution. Microperfusion with a Ca2(+)-free Ringer containing Blue Dextran did not affect the miniature endplate potential frequency or amplitude. Changes of spontaneous transmitter release were studied either during microperfusion of Ringer containing 5 mM Ca2+ or during microperfusion of 2 mM Ca2+ simultaneously with the stimulation of the motor nerve. This second procedure also permitted study of the characteristics of evoked release. Microperfusion of Ca2+ induced a larger and more rapid increase in the miniature endplate potential frequency in proximal than in distal regions. The time required for the miniature endplate potential frequency to return to the control value after Ca2+ microperfusion was longer than the time needed to increase the frequency and this decay period was longer in the proximal region than in the distal one. Moreover, miniature endplate potentials produced in proximal regions, were typically larger and more variable than those produced in distal regions. In five experiments, the endplate potentials produced by 100-200 pulse pairs (interval of 15 ms at every 2 s) were recorded intracellularly during the microperfusion. The quantal content of the first endplate potential of the pair (EPP1) was systematically smaller in distal regions than in proximal regions. The percentage of failures and the coefficients of variation were higher in distal than in proximal regions, indicating a larger variability of quantal content. The frequency facilitation was not different between the two regions, but, however, the second stimuli of the pair usually produced a net increase of transmitter release which was greater in proximal than distal regions. Our experiments demonstrate that both the spontaneous and the evoked release are more responsive to Ca2+ application in the proximal than in the distal regions of the frog neuromuscular junction.

A prion protein variant in a family with the telencephalic form of Gerstmann-Sträussler-Scheinker syndrome. We present a patient with a mutation in the open reading frame of the prion protein gene (PRNP), which results in substitution of valine for alanine at codon 117. The patient is a member of a large American kindred of German descent with the telencephalic form of Gerstmann-Sträussler-Scheinker syndrome (GSS). Two other affected members of this kindred carried this mutation, as inferred from haplotypes of their offspring and spouses. The mutation was absent in one member with a protracted neurologic illness that differed from the other affected members' illnesses. The identification of a distinct PRNP mutation in the telencephalic form of GSS supports the hypothesis that allelic forms of PRNP may correspond to distinct clinical disease entities.

Cholecystokinin and somatostatin in Alzheimer's disease postmortem cerebral cortex. The neuropeptides cholecystokinin (CCK) and somatostatin are synthesized in separate, but morphologically similar, populations of locally projecting neurons in cerebral cortex. Concentrations of somatostatin are markedly diminished in Alzheimer's disease (AD), suggesting possible dysfunction of the intrinsic cortical neurons in which it is produced. We determined whether cortical levels of CCK might be similarly affected in AD by dissecting postmortem brain samples from 14 histologically confirmed cases of AD and 17 age-matched controls and measuring CCK and somatostatin by radioimmunoassay. CCK-like immunoreactivity was significantly reduced in the AD brains by 24 to 38% in five of the 11 cortical areas examined but was normal in the remaining regions. Somatostatin concentrations measured in the same tissue extracts were consistently decreased by 45 to 65%. These results indicate that (1) CCK-immunoreactive neurons in cortex are affected by the AD process, and (2) the changes in levels of CCK are less dramatic than the reductions of somatostatin immunoreactivity.

Linkage analysis of juvenile parkinsonism to tyrosine hydroxylase gene locus on chromosome 11. We performed linkage analyses of juvenile parkinsonism with autosomal recessive inheritance (AR-JP) to chromosome 11p15.5 locus including tyrosine hydroxylase (TH), Harvey-ras-1 (HRAS), and insulin (INS) genes by analyzing genomic DNA from 28 members of seven Japanese AR-JP families containing 10 affected individuals. We used the methods of pairwise linkage analysis (LIPED) and multipoint linkage analysis (LINKAGE) for genetic linkage to the chromosome 11p15.5 markers and detected recombination events between AR-JP locus and TH gene. Multipoint linkage analysis excluded the linkage in the interval between TH and HRAS, as well as 11 cMo distal to TH and 8.5 cMo distal to HRAS, a total of approximately 23 cMo on chromosome 11p.

2-Amino-3-phosphonopropionate blocks the induction and maintenance of long-term potentiation in rat hippocampal slices. Rat hippocampal slices were used to examine the effects of 2-amino-3-phosphonopropionate (AP3), an inhibitor of phosphatidylinositol (PI) turnover linked to metabotropic quisqualate receptors, on the development and maintenance of long-term potentiation (LTP) in area CA1. When perfused for 5 min prior to tetanization at concentrations of 100-1000 microM, D,L-AP3 had no effect on baseline synaptic transmission but blocked posttetanic potentiation (PTP) and the induction of LTP. Unlike the N-methyl-D-aspartate (NMDA) antagonists, 2-amino-5-phosphonovalerate (AP5) and MK-801, AP3 eliminated the late phase of LTP when applied immediately after tetanization. These data support the hypothesis that PI turnover is a factor in the expression and maintenance of LTP.

Striatal glutamic acid decarboxylase immunoreactivity is increased after dopaminergic deafferentation: densitometric analysis. Several lines of evidence suggest that dopamine exerts a chronic inhibitory action on GABAergic cells in the striatum, and striatal glutamic acid decarboxylase (GAD) mRNA levels are increased after ipsilateral dopaminergic denervation. In the present study we have used GAD immunocytochemistry to assess whether dopaminergic denervation results in an increase in GAD protein synthesis. In three 6-hydroxydopamine-lesioned animals, there was a perceptible increase in the density of GAD-immunoreactive (ir)staining on the side ipsilateral to the lesion. Computer-assisted densitometric analysis showed a significant increase in GAD-ir staining in the ipsilateral striatum compared to the contralateral (control) side. These data suggest that removal of striatal dopaminergic innervation results in an increase in the amount of immunoreactive GAD, the rate limiting enzyme in the synthesis of GABA.

Distribution of somatostatin- and neuropeptide Y-immunoreactive nerve fibers in the porcine female reproductive system. The localization and distribution of somatostatin and neuropeptide Y were studied in the porcine female reproductive system with the indirect immunofluorescence technique. Somatostatin-immunoreactive nerve fibers were observed in different parts of the ovary and in the muscular membrane of the uterus as well as in the mesosalphinx. Somatostatin-immunoreactive neurons were detected in the inferior mesenteric ganglion. Neuropeptide Y immunoreactivity was present in a large number of nerve fibers distributed in different regions of the uterus, oviduct and ovary. The present results suggest that the porcine female genital organs receive innervation by somatostatin- and neuropeptide Y-containing nerve fibers, but their exact functional role remains to be established.

Increase of dopamine beta-hydroxylase immunoreactivity in non-noradrenergic nerves of rat cerebral arteries following long-term sympathectomy. The expression of dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) immunoreactivity (IR) after short-term (2 days) and long-term (3 weeks) sympathectomy was investigated in rat cerebral vessels, dura mater and pterygopalatine ganglion neurones (which are known to project to cerebral arteries) by immunohistochemistry at both the light and electron microscopical levels. TH-IR, like glyoxylic acid-induced fluorescence, was completely abolished by sympathectomy. By contrast, DBH-IR was localized in nerve fibres, lacking 5-hydroxydopamine (5-OHDA)-labelled vesicles, along cerebral vessels of long-term sympathectomized rats, but not in the dura mater, and in pterygopalatine ganglia, where the number of DBH-IR neurons increased from 27.87% to 54.11%. Since virtually all the pterygopalatine neurons displayed choline acetyltransferase (ChAT)-IR, both in control and sympathectomized rats, it is concluded that long-term sympathectomy caused an increase of the expression of DBH-IR in cholinergic neurones of the pterygopalatine ganglion, without these neurons producing or storing noradrenaline.

Coexistence of glutamate and choline acetyltransferase in a major subpopulation of laryngeal motoneurons of the rat. Glutamate immunoreactivity was found in 88% of the laryngeal motoneurons which were located in the ambiguus nucleus and the retrofacial nucleus. The glutamate-containing laryngeal motoneurons were identified by double labeling using the retrograde tracing of True blue from the cut end of the recurrent laryngeal nerve and immunocytochemistry of glutamate. On the other hand, choline acetyltransferase (ChAT) immunoreactivity was found in all the laryngeal motoneurons which were similarly identified as the origin of the recurrent laryngeal nerve using retrograde tracing of True blue. Since glutamate coexists richly with ChAT in a major subpopulation of laryngeal motoneurons, it is probable that glutamate may play a role as a co-transmitter in the cholinergic motoneurons.