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Directly following transfer to in vitro conditions, lumbar dorsal root ganglia (DRG) from triiodothyronine (T3)-treated premetamorphic tadpoles showed an approximately three fold increase in incorporation of bath-applied radiolabeled amino acid relative to DRG from vehicle-treated tadpoles. By contrast, at 1 h after transfer, no increased incorporation was detected. At this time, examination of in-vitro-synthesized [35S]proteins by two-dimensional gel electrophoresis revealed a selective enhancement of a slightly acidic 25-kilodalton (kD) polypeptide. Addition of 1 M glycerol to the incubation medium prevented the enhanced synthesis of the low-molecular-weight polypeptide and preserved the T3 response. When DRG were exposed in situ, the T3 response - monitored following intraganglionic injection of [35S]methionine-was detected after 2 h, but not after 4 h. Four hours were also the earliest time at which an elevated level of the 25-kD polypeptide was observed. The correlation between loss of hormonal responsiveness and appearance of a putative stress protein is of general importance as a cautionary note for in vitro studies of endocrine effects on developing tissues.

The immunocytochemical staining of tyrosylated alpha-tubulin using the specific monoclonal antibody YL 1/2 decreases dramatically in the parallel fibers of the rat cerebellum during development. Detyrosylation starts at 14 days in the deeper part of the molecular layer and then spreads over the entire molecular layer within a week. Thyroid deficiency induces a marked delay in alpha-tubulin detyrosylation in the parallel fibers. Detyrosylation begins only at the age of 28 days in hypothyroid animals. Immunobinding assays of detyrosylated and tyrosylated alpha-tubulin concentrations in the cerebellar supernatant of developing rats confirm that thyroid deficiency increases the tyrosylated alpha-tubulin content at 21 and 35 days. Short-term thyroxine treatment (daily from day 8, 10 or 12, to day 14) do not induce the normal pattern of tubulin detyrosylation starting at 14 days, although they do induce parallel fiber lengthening. Daily treatment from birth is required to produce the alpha-tubulin modification found in normal rats. We propose that the microtubule detyrosylation which occurs during axonal maturation is not a prerequisite for axon growth and synaptic differentiation, but may rather be implicated in the stabilization of microtubules in the mature axons.

The expression of NF-H neurofilament subunit mRNAs was investigated in the rat brain at different ontogenic stages. The levels of NF-H mRNAs vary 15-fold among brain regions with the highest level in the brainstem. In situ localization studies revealed that the NF-H mRNAs are mainly concentrated in the brainstem motoneuron nuclei. By increasing the sensitivity of the hybridization method, NF-H mRNAs could also be localized in neurons present in the cortex, thalamus and hippocampus areas. Minor amounts of NF-H mRNAs were already detected at 17-day embryonic stages.

The adenomatoid odontogenic tumour (AOT) is a rare benign neoplasm of odontogenic origin. A review of the available published literature reports on AOT from Africa reveals that 33 cases appear to have been reported and that the tumour tends to occur in young patients, from 10-28 years with a mean age of 16.2 years. Females are affected more than males, with a female:male ratio of 3:2. 65.6% of cases were in the maxilla and 34.4% in the mandible, the majority of which (92.3%) are located in the anterior region of the jaws. 66.7% of the cases are associated with unerupted teeth. Clinically, AOT shows increasingly painless swellings which may eventually cause facial deformity. Tentative clinical diagnosis based on radiology is not pathognomonic for AOT; histopathological confirmation is necessary since incorrect diagnosis may lead to mutilating surgery whereas the lesion is benign with limited growth potential and treatment requires only simple surgical enucleation. Recurrence is rare.

A study of 59 patients with middle third maxillofacial fractures over a 5 year period was undertaken. The incidence of, pattern of fracture, type of accompanying injuries, and assessment of the outcome of given treatment were studied. The young adult male featured prominently in the study giving a male/female ratio of 14:1. The zygomatico-maxillary complex was fractured in 42.4% of the cases, while the Le Fort 1 fracture was the most common of the Le Fort Fracture types. Road traffic accidents accounted for 81.4% of the aetiological factors, while armed robbery attacks was the next common source of trauma at 6.8%. Treatment by the Gillies temporal approach and immobilization within the tissues techniques produced very satisfactory results in 96% of the patients who received treatment. Loss of vision in one eye was the most common residual complication.

This study was conducted during 1987-1988 academic year in the rural areas of Tihama Saudi Arabia to assess the average duration of breast feeding and the effect of some factors. A multi-way analysis of variance approach was used to examine the effect of mother's age, parity and education on the duration of breast feeding. The mean duration of breast feeding was 11.2 months +/- and the results of the regression analysis shows all the three maternal variables, age, parity and education to have statistically significant independent effect on the duration of breast feeding. The results showed that 98.3% support breast feeding and 78.9% of the sample were illiterates. These findings are discussed in relation to previous work.

During 1987-1988, paramedical personnel interviewed 923 women who have attend the primary health care center in the Tiahama Valleys in mountainous southwestern Saudi Arabia to learn how long women breast feed and what factors affect its duration. 78.9% of the women were illiterate as compared to 50% for the total rural population of mothers in Saudi Arabia. 90% had 1 child. 98.35% believed in the importance of breast feeding, yet only 50.7% breast fed their infants. 47.3% mixed fed and 2% bottle fed their infants. 35% began solid foods (cereals, rice, eggs, and vegetables) when the child was 6 months old. Age of the mother had a statistically significant positive effect on the duration of breast feeding (p.05). Indeed mothers 25 years old were more likely to breast feed for 6 months than their older counterparts. Nevertheless these women did have an average duration of breast feeding of 9.3 months whereas the older mothers breast fed an average of 11 months. Parity also significantly affected duration of breast feeding in a positive manner (p.01). For example, 33% of grand multiparous mothers did not breast feed for 6 months compared to 66% of the primigravidas. Education had a significant negative effect (p.01). In fact, 68% of mothers with university level education breast fed for 6 months or less. The average duration of breast feeding stood at 11.2 months (median, 10 months; mode, 12 months) while the average of all rural areas in Saudi Arabia in 1988 stood at 14.2 months. Completion of weaning occurred on average at 11.7 months. Mothers who only bottle fed did so for an average of 11.4 months. These results indicate a need for health care teams to promote breast feeding in this area.

In a study by questionnaire and anthropometric measurements of the effect of sickle cell disease on the health growth and education of 102 Nigerian children aged between 9 months and 17 years, the first symptoms of the disease had occurred by the age 1 1/2 years in 58.8% of them. Strenuous exercise and exposure to cold water and weather commonly precipitated illness episodes which occurred in 81.4% of the children at least bi-annually. 94.1% of the children have been hospitalised and 76.5% transfused with blood at least once each before. Of the 67 children in school only 32.8% were in their correct classes and 53.7% have lost years. The mean class examination performance score computed for 55 of those in school was 67.8% +/- 21.5. The heights fall around and the weights below the third percentile of standard growth curves for Nigerian elite children. The disease affects the parameters examined adversely and should be controlled more effectively.

Primary cerebral non-Hodgkin's lymphoma of the brain are very rare neoplasms. The pertinent data describing these tumours have been gathered in retrospect and from reviews of postmortem materials. There has been increased interest in this disorder in recent years because of improved diagnostic capabilities. The computerized axial tomography and radiopharmaceutical uptake brain scintiscans are invaluable prebiopsy non-invasive investigative procedures. Brain biopsy and minimal tumour resection are nowadays the preferred modes of surgical treatment. Because more radical surgery and whole brain radiation have failed to control the high rate of systemic relapses, it is generally recommended that Chemotherapy should be incorporated into the primary treatment plan. High dose intravenous methotrexate has been shown to be effective in treatment of residual lymphoma and systemic recurrent tumours.

In this study, a total of 519 patients were interviewed. 82.5% had incomplete abortion. The implication of abortion especially when induced is emphasised. Economic implications that are contributed by the youth are stressed. 83.6% of the patients had not used any contraception. The role of contraception in preventing unwanted pregnancy and therefore induced abortion is stressed. The role of the physician in providing contraception and appropriate contraceptive knowledge is discussed.

A study of 519 consecutive women admitted to Kenyatta National Hospital with the diagnosis of abortion revealed that the majority were young and had a history of nonuse of contraception. Abortion was incomplete in 428 (83%) of cases; 60 (12%) cases involved sepsis. Women 20-24 years of age accounted for 221 (43%) of the abortions; the other two most represented age groups were 25-29 years (28%) and 14-19 years (17%). 460 (89%) of the abortion patients had never used a contraceptive method. The most frequently cited reasons for nonuse were desire for pregnancy (48%), no conscious reason (13%), procrastination in getting to a family planning clinic (8%), no knowledge of family planning (6%), and fear of side effects (6%). Of the 64 cases of failed contraception, 27 were using the pill, 25 had an IUD in place, and 8 were relying on the rhythm method. Among contraceptive users, the major sources of information about contraception were nurses (52%), radio and newspapers (19%), and other women (15%). Only 4% indicated that a physician had discussed family planning with them. Given the resource drain that treatment of incomplete abortion can place on Kenya's health care system and the risk of abortion-induced pelvic infection and subsequent infertility, Kenya's health workers should be encouraged to be more aggressive in promoting family planning use among young women.

One hundred consecutive patients with diabetic ulcers were studied in an 8-month-period. There were 58 females. The mean age was 59.9 years. Eighty three patients had non-insulin dependent diabetes mellitus. The mean duration of diabetes mellitus was 11.6 years. The mean duration of the ulcer was 8.5 months. Sixty nine of the ulcers were gangrenous. Over 50% of the ulcers involved the big toes. Neuropathic ulcers were found mainly in the sole of the feet. Roentgenograms showed evidence of osteomyelitis in 44 patients. There were 356 bacterial isolates (340 aerobes and 16 anaerobes) from the ulcers. There were 3.6 infecting organisms per ulcer in gangrenous ulcers, while in neuropathic ulcers, there were 3.4 infecting organisms per ulcer. In both types of ulcer Staphylococcus aureus and Escherichia coli were the commonest infecting organisms each being isolated in 88 of the 100 ulcers studied. In repeat bacterial cultures at 4 weeks there were 116 bacterial isolates. Staphylococcus aureus persisted in 63 ulcers despite therapy, while Escherichia coli persisted in 35. There were no new organisms isolated at repeat cultures and no ulcer was completely sterile. The Staphylococcus aureus was 100% sensitive to Augmentin (Amoxicillin plus clavulinic acid), Clindamycin, Novobiocin, and Amikacin while the gram negative bacilli were sensitive to Cefotaxime, Piperacillin, Amikacin and augmentin, Clindamycin, Chloramphenicol and Lincomycin inhibited the growth of anaerobes to a varying degree.

Acute and chronic renal failure (ARF and CRF) are primary health problems in health centres, district and provincial hospitals. Their managements should be initiated in these areas of the health services. Some of the managements of CRF & ARF should be initiated in private clinics by private practitioners. ARF is a medical emergency while CRF is insidious with non-specific features. Discussions on CRF and ARF and their timely managements are mandatory if the mortality and morbidity associated with them are to be prevented.

We tested 120 consecutive admissions with sputum positive pulmonary tuberculosis for antibodies to the human immunodeficiency virus type I (HIV-1). Pre-treatment chest x-ray appearances were recorded. Seventy one patients (59%) were males and 49 (41%) were females. 43 (35.8%) patients were seropositive for HIV, and were all in the age range 16-45 years. The seropositivity for the 56 males and 44 females in the age range 16-45 years were 53 and 26 percent respectively. Atypical x-ray features were found in 21 of 43 cases compared to 15 of 57 referents (p less than 0.025). Radiographic features typical of reactivation pulmonary tuberculosis in the adult were found in 73% and 51% of referents and cases respectively (P less than 0.025). Pulmonary lesions localized to the mid and or lower zones were seen in 20 percent of cases and 3.3% of referents (P = 0.01). Mediastinal and or hilar adenopathy alone or with pulmonary infiltrates occurred more frequently among cases but the results were not significant. Our findings indicate that radiological appearances of pulmonary tuberculosis in patients seropositive for HIV-1 antibodies tend to be atypical in type of lesion and or anatomic distribution, even for patients from communities with high prevalence rates of tuberculosis.

The authors tested 120 consecutive admissions with sputum positive pulmonary tuberculosis for antibodies to the human immunodeficiency virus type I (HIV-1). Pretreatment chest x-ray appearances were recorded. 71 patients (59%) were males and 49 (41.9%) were females. 43 (35.8%) patients were seropositive for HIV, and were all between the ages of 16-45. The seropositivity for the 56 males and 44 females in this age ranger were 53% and 26%, respectively. Atypical x-ray features were found in 21 of 43 cases compared to 15 of 5 referents (p 0.25). Radiographic features typical of reactivation pulmonary tuberculosis in the adult were found in 73% and 51% of the referents and cases, respectively (p0.025). Pulmonary lesions localized to the middle or lower zones were seen in 20% of cases and 3.3% of the referents (p=0.01). Mediastinal or hilar adenopathy alone or with pulmonary infiltrates occurred more frequently in cases but the results were not significant. These findings indicate that radiological appearances of pulmonary tuberculosis in patients positive for HIV-1 antibodies tend to be atypical in type of lesion and/or anatomic distribution, even for those patients from communities with high prevalence rates of tuberculosis.

A report is presented of a 45 day old female infant with congenital amputation of the great toe, complete constriction bands of the lower leg and fingers all of which are explained by congenital amniotic band syndrome. There is a brief review of the literature on the pathology and possible aetiology.

Only recently has attention turned to the needs of children in the EMS system, and it has been shown that there is work to be done if these needs are to be met. The founders of EMS systems were trained in adult specialties and worked without input from the pediatric community. It is not surprising that the special needs of children within an adult-oriented EMS system were underemphasized. Children make up less than 10% of prehospital runs and less than 5% of the critically ill patients. Numerous EMS systems nationwide are undertaking this work and federal support is evident through the Maternal and Child Health EMSC program. Vital issues include the need for experts in emergency medical services to work together with experts in pediatric emergency care and for sound program evaluations to be performed to demonstrate the efficacy of these new programs.

In-hospital care of seriously ill children has improved dramatically with advances in emergency medicine and pediatric intensive care. Interhospital transport is necessary for a large percentage of critically ill or injured children. The appropriateness of care received during the transport phase is a crucial component of the critical care continuum. There are few widely applicable or accepted standards in place for pediatric transport, partly because standards are in the developmental phase and partly because of the lack of uniformity of resources available in different geographical regions. A common sense approach to providing appropriate patient care should be used, keeping in mind the philosophic and practical considerations detailed above. The best interest of the individual patient should be the primary factor in making any transport decision.

Accurate patient triage to provide early identification of potentially seriously ill or high-risk infants and children is an important part of any emergency care system. Use of the SAVE-A-CHILD mnemonic in a busy ED setting provides systematic organization of important clinical observations that may serve as markers of serious disease. Early recognition of the high-risk patient will reduce morbidity and mortality. The discussion included may be helpful to emergency physicians in training their staff to provide a safe triage environment.

Recognition of the high-risk mother and fetus is an essential component of resuscitation if it is to be organized, accurate, and successful. Although many high-risk factors put the neonate in jeopardy, the first responder or ED physician can plan the initial approach to resuscitation by knowing the answer to three questions: (1) Is there particulate meconium in the amniotic fluid? (2) Is the fetus/baby premature? and (3) Is a multiple gestation pregnancy expected? Although the ABCs (airway, breathing, circulation, chemical) of resuscitation adequately describe the parameters essential for adequate resuscitation, they do not relate to the actions necessary to accomplish this feat; therefore, it is important for participants in neonatal resuscitation to remember the acronym SOS--suction, oxygen, stimulation. Even infants with moderate depression (occasional respiratory effort) will generally respond to brief suctioning of the airway, 100% oxygen, and vigorous stimulation. If SOS is ineffective, PPV must be administered without hesitation. Resuscitation of the newborn is not a simple procedure, but rather a dynamic process involving continuous evaluation, action, and reassessment before, during, and after the actual resuscitation. A specially trained resuscitation team of physician, nurse, and respiratory therapist interacting in a dynamic evaluation approach is ideal but not practical for most community hospitals. Each hospital must develop its own protocol and train and maintain its resuscitation personnel. If each member of the team is prepared to take over in the absence of the others, neonatal resuscitation will remain fast, organized, and accurate.

Effective evaluation and management of the pediatric trauma patient is based on knowledge of the unique anatomic and pathophysiologic differences in children. An understanding of these differences along with the trauma resuscitation guidelines established by the American College of Surgeons will allow the trauma team to provide systematic and comprehensive resuscitation of the child with multiple injuries. Continued research in the field of pediatric trauma resuscitation and the ongoing efforts of the National Pediatric Trauma Registry will continue to advance our understanding and management of injured children.

The role of the emergency physician in optimizing outcome for the maternal and fetal victims of trauma is pivotal. Knowledge of the anatomic and physiologic changes of pregnancy aid in understanding the nuances of care of the pregnant trauma patient. Both catastrophic and noncatastrophic trauma can be managed with confidence and expertise by recalling the maternal and fetal pathophysiologic responses to trauma. Burns and electrical injuries carry significant fetal risks, which may be minimized by rapid and knowledgeable emergency care.

Dehydration caused by diarrhea remains a major source of morbidity and mortality worldwide. Dehydration is a common clinical presentation seen by most physicians. Clinical diagnosis depends on the recognition of signs and symptoms as well as change in weight. Laboratory studies are helpful in categorizing the dehydration as isotonic, hyponatremic, or hypernatremic, which is necessary to plan appropriate therapy. In many situations, oral rehydration therapy is possible and desirable. Intravenous rehydration remains the standard of care for children with severe dehydration and shock.

Clinical conditions that require operative intervention are frequent causes of emergency department visits. Common causes such as pyloric stenosis, intussusception, inguinal hernias, torsion of the testicle, and ingested foreign bodies are discussed, and aids in diagnosis and early intervention are addressed. Conditions that mimic surgical problems also are mentioned.

A rapid controlled induction of anesthesia is useful to facilitate emergency intubation and to reduce the complications of intubation in pediatric patients. A protocol for rapid sequence intubation and suggestions for optimizing airway management in the Emergency Department are described. The use of end tidal carbon dioxide monitoring and pulse oximetry are strongly advocated to monitor all intubations in the Emergency Department.

Pediatric procedures in the Emergency Department typically involve techniques for stabilization, evaluation, and treatment of the child. This article considers indications and techniques for vascular access, lumbar puncture, arterial lines, pulse oximetry, and urine collection. Multiple routes of medication administration and pain management modalities also are reviewed.

A thorough understanding of the spectrum and ramifications of pediatric HIV infection is important to the emergency physician. Rising reports of cases in rural populations and the vertical transmission from mother to infant are important changes in HIV epidemiology. Nearly all organ systems are either directly or indirectly involved and problems encountered, including diagnostic and therapeutic considerations by organ system, are reviewed. The role of the emergency physician in caring for HIV-infected infants and children includes recognition, timely treatment of complications, protection of self and other health care workers, and protection of the immunocompromised child.

Observation units for children in an Emergency Department setting can serve to improve the quality of medical care provided as well as reduce overall costs; however, they must be properly organized with careful consideration for the needs of children. Policies must be written specifying who is in charge as well as who can be accepted into these units and for how long. Procedures regarding documentation and sign-out must be formulated. These units must be well staffed and fully equipped, and they should be pleasant places for the children to stay; otherwise, what might begin as assets can quickly become disorganized and potentially dangerous liabilities.

The process of providing emergency care is a difficult one. In pediatric emergency care, this process is complicated by a host of factors, including special legal considerations. A basic identification of these legal issues as discussed will enhance the ability of the emergency physician to feel more comfortable in an already complicated work environment. More important, a working knowledge of legal principles allows additional time during a crisis to focus on quality medicine rather than ponder legal considerations. When questions of law versus medicine conflict, it is best to consult competent legal authority. When time does not permit such access, it is imperative that proper medical care is pursued to the best ability of the physician provider. No matter what the legal outcome in a particular case, one must feel comfortable within professional practice standards and be able to live with oneself.

There is considerable, although not entirely consistent, evidence that the hippocampus inhibits most aspects of HPA activity, including basal (circadian nadir) and circadian peak secretion as well as the onset and termination of responses to stress. Although much of the evidence for these effects rests only on the measurement of corticosteroids, recent lesion and implant studies indicate that the hippocampus regulates adrenocortical activity at the hypothalamic level, via the expression and secretion of ACTH secretagogues. Such inhibition results largely from the mediation of corticosteroid feedback, although more work is required to determine whether the hippocampus supplies a tonic inhibitory input in the absence of corticosteroids. It must be noted that the hippocampus is not the only feedback site in the adrenocortical system, since removal of its input only reduces, but does not abolish, the efficacy of corticosteroid inhibition, and since other elements of the axis appear eventually to compensate for deficits in feedback regulation. The importance of other feedback sites is further suggested not only by the presence of corticosteroid receptors in other parts of the brain and pituitary, but also by the improved prediction of CRF levels by combined hypothalamic and hippocampal receptor occupancy. The likelihood of feedback mediated by nonhippocampal sites underscores the need for future work to characterize hippocampal influence on HPA activity in the absence of changes in corticosteroid secretion. However, despite the fact that the hippocampus is not the only feedback site, it is distinguished from most potential feedback sites, including the hypothalamus and pituitary, by its high content of both type I and II corticosteroid receptors. The hippocampus is therefore capable of mediating inhibition over a wide range of steroid levels. The low end of this range is represented by corticosteroid inhibition of basal (circadian nadir) HPA activity. The apparent type I receptor specificity of this inhibition and the elevation of trough corticosteroid levels after hippocampal damage support a role for hippocampal type I receptors in regulating basal HPA activity. It is possible that basal activity is controlled in part through hippocampal inhibition of vasopressin, since the inhibition of portal blood vasopressin correlates with lower levels of hippocampal receptor occupancy, and the expression of vasopressin by some CRF neurons is sensitive to very low corticosteroid levels. At the high end of the physiological range, stress-induced or circadian peak corticosteroid secretion correlates strongly with occupancy of the lower affinity hippocampal type II receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

With the understanding that various drugs, industrial chemicals, and chemicals of environmental importance can increase thyroid hormone hepatic metabolism and excretion, it is important to consider whether compensation by the thyroid gland for this increased excretion can lead to stimulation of the hypothalamic-pituitary-thyroid axis and possibly secondary hyperplastic or neoplastic changes in the thyroid. The compounds discussed in this review all affect thyroid function by increasing biliary excretion of thyroid hormone metabolites. Numerous studies have been performed to elucidate the effect of these drugs on thyroid hormone equilibrium. Animals given PB compensate for the increased biliary excretion with an elevated TSH allowing maintenance of a euthyroid state. Human studies demonstrate increased thyroid hormone plasma clearance, but without an increased TSH. The effects of an experimental leukotriene antagonist are similar. In humans, diphenylhydantoin has been conclusively shown to cause a decrease in peripheral thyroid hormone levels, although without evident hypothyroidism or increase in TSH. Limited studies of rifampin and carbamazepine reveal similar results. Nicardipine in rats causes reproducible decreases in free T4 levels, although it does not clearly stimulate a rise in TSH levels. An experimental imidazole caused reversible lowering of peripheral thyroid hormone levels in rats; in this study TSH was not measured. Studies with aromatic hydrocarbons administered to rats reveal a general decrease in T4 levels, with a compensatory increase of TSH. The effects of chronic administration of a compound that can cause increased thyroid hormone metabolism with compensation via increased TSH production are of more than theoretical interest, as it has been well documented that constant stimulation of the thyroid gland in rats with supraphysiological levels of TSH causes goiter, thyroid hyperplasia, adenomas, and carcinomas. In humans with congenital metabolic thyroid deficiencies there is an increased incidence of thyroid neoplasia, suggesting an association with chronic increased TSH levels. In contrast, however, large epidemiological studies of areas of endemic goiter do not show an association of human thyroid cancer with iodine deficiency and presumed chronic thyroid gland stimulation. Histological evidence of thyroid follicular hyperplasia has been noted in rats after administration of phenobarbital, nicardipine, polycyclic hydrocarbons, and possibly an experimental imidazole. Increased thyroid gland size has been demonstrated in rats given phenobarbital, nicardipine, a leukotriene antagonist, and various polycyclic hydrocarbons. Thyroid carcinoma in rats has been associated with treatment with nicardipine and after exposure to several aromatic hydrocarbons.(ABSTRACT TRUNCATED AT 400 WORDS)

CgA is a 49 kilodalton protein that is present in the secretory granules of most endocrine and many neuroendocrine cells. Detection of CgA in cells by immunocytochemistry and measurement of CgA in serum by immunoassay can serve as tissue and serum markers for CgA-producing tumors. CgA is of diagnostic value in classical endocrine tumors, in hormone-negative tumors, and in endocrine tumors in which other diagnostic procedures have their limitations. Although the biological function of CgA is yet unknown, it may serve as a precursor molecule, like POMC, for a family of biologically active peptides. CgA is an important new tool for the endocrinologist in the diagnosis and management of patients with endocrine and neuroendocrine tumors.

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.

In a preceding paper we reported on the detection and characterization of human serum amyloid A protein (SAA) in very low density lipoproteins (VLDL) and high density lipoproteins (HDL) of patients after acute myocardial infarction. Here we describe the time course of the occurrence of SAA in VLDL and HDL in the postinfarction period. SAA reached its maximum in VLDL and HDL approximately 53 h after the acute event. At the peak of the acute-phase response, SAA comprised as much as 38% of the total apoproteins of VLDL and HDL. SAA appeared at the same points in time and with nearly the same concentrations in VLDL and HDL. We conclude that SAA is not exchanged in plasma between lipoproteins of different densities and that this protein is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle.

The influence of carbohydrate moieties of transferrin (Tf) on the determination of its molecular mass (MM) by polyacrylamide gradient gel electrophoresis (PAGGE) was investigated. Iron-free native human serum transferrin (Tf) of 99% purity and partly or completely carbohydrate- and N-acetylneuraminic acid (NANA)-free molecule forms were analyzed. The MM differences before and after enzymatic cleavage were found not to agree with the theoretical difference. From amino acid and carbohydrate analysis the MM of Tf was determined to be 79,570 Da whereas by denaturing and nondenaturing PAGGE MM of 77,000 Da +/- 1000 Da were found. After enzymatic cleavage of the two carbohydrate chains of Tf the difference between the calculated MM and the value reported in literature increased to 7000 Da (nondenaturing PAGGE) and 9200 Da (denaturing PAGGE). Following enzymatic cleavage of the 4 NANA molecules (MM 1237 Da) we obtained the relatively largest difference between the value given in the literature and that determined by PAGGE, namely MM 3300 Da on nondenaturing and 4000 Da on denaturing PAGGE. The differences due to the removal of the other carbohydrates were negligible. In addition we tested the periodic acid-Schiff reagent to stain iron-free Tf, containing different carbohydrate residues. The shortest carbohydrate moiety necessary for Tf staining corresponds to two identical carbohydrate chains of the structure (Asn)-GlcNAc-GlcNAc-beta-Man-(alpha-Man-)-alpha-Man.

In the absence of vitamin K or in the presence of the vitamin K antagonists, abnormal nonfunctional forms of prothrombin circulate in the blood. A reliable and reproducible technique, derived from traditional crossed affinoimmunoelectrophoresis in presence of calcium lactate, was developed and optimized. The technique is based on nondenaturing polyacrylamide gel affinoelectrophoresis, with calcium lactate, of plasma samples, followed by immunoblotting with rabbit anti-human prothrombin serum and detection with an anti-rabbit immunoglobulin peroxidase conjugate. Depending on the plasmas, one or two bands were visualized and quantified by densitometry of the immunoblots. The technique was able to detect abnormal des-gamma-carboxylated prothrombins at concentration of 0.1 microgram/mL.

Homogeneous (7.5%) and gradient (10-15%) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98,000 and 96,000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93,000, 93,000 and 92,000), respectively, on the other. In the native gels the enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raised against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.

A facile two-dimensional gel electrophoresis procedure has been developed for the analysis of neural tissue proteins which eliminates the serious problems associated with protein insolubility at the point of sample application onto polymerized first-dimension isoelectric focusing gels. This was accomplished by combining the methods of two previously published procedures. Our procedure provides an alternative method to the complex gel systems often employed for less soluble proteins, and yields very reproducible, high resolution separations. This procedure, which is in routine use in our laboratories for the analysis of total proteins extracted from retina and brain, produces protein patterns that are easily compared using both visual and computer-assisted image analysis techniques. Presented here are the results of a set of experiments designed to identify proteins unique to retina. This procedure should be useful to investigators studying protein changes resulting from genetic mutation, development, drug treatment or disease, in neural tissue as well as in virtually all other tissues.

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a = 5.262(6) nm, b = 7.631(8) nm, c = 3.945(5) nm and beta = 94.47(9) degrees. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T.A., Bergfors, T., Sedzik, J. & Unge, T. (1988) EMBO J. 7, 1597-1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel beta strands forming a beta barrel, and by two alpha helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.

A hybrid peptide of cecropin A and melittin was investigated by two-dimensional 1H-NMR at pH 5.8 in aqueous solution with 30% (by vol.) hexafluoroisopropanol. The peptide contains 26 amino acids, is a combination of the first 13 residues of each of the two parent peptides, CA(1-13)M(1-13) identical to CAM(1-26) and has an amidated C terminal. This peptide was recently synthesized [Boman, H.G., Wade, D., Boman, I.A., Wåhlin, B. & Merrifield, R.B. (1989) FEBS Lett. 259, 103-106] and shown to have strong antibacterial activity but to be harmless towards erythrocytes. All resonances of the main chain and side chain beta-protons are assigned except for those of the N-terminal lysine. Several medium range NOE connectivities were observed showing two separated alpha-helices, involving residues 4-12 and 16-26. The JNH alpha-coupling constants in these sections support the conclusion. From the exchange rates of the NH protons it is concluded that the alpha-helix of residues 16-26 is much more stable than the other helix. The circular dichroism data indicates about 30% less alpha-helix character than the NMR data. A reduced contribution to the ellipticity from the unstable helix is suggested. The chemical-shift differences between the two parts of the hybrid and the respective parent peptides are larger for the cecropin part than for the melittin part. For the latter, residues 17-26 of the hybrid are proposed to have a secondary structure very similar to that of residues 4-13 of melittin.(ABSTRACT TRUNCATED AT 250 WORDS)

A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn- Pro-Ser-Pro10-Asp-Ala-Ser-Pro-Glu-Leu-Ser-Lys-Tyr20-Met-Leu- Ala-Val-Arg-Asn- Tyr-Ile-Asn-Leu30-Ile-Thr-Arg-Gln-Arg-Tyr CONH2. This sequence shows stronger structural similarity with pig neuropeptide Y (64%), particularly in the COOH-terminal region, than with pig peptide tyrosine--tyrosine (61%) or with pig pancreatic polypeptide (42%). Molecular modelling and dynamic simulation, based upon sequence similarity with turkey pancreatic polypeptide, indicates that the conformations of the polyproline-helix-like region (residues 1-8) and the alpha-helical region (residues 15-30) in turkey pancreatic polypeptide are conserved in peptide methionine-tyrosine, and that non-bonded interactions between these domains have preserved the overall polypeptide fold in the molecule. The substitution of the otherwise totally conserved Gly9 residue by serine in lamprey peptide methionine-tyrosine, however, results in a preferred structure in which the conformation of the beta-turn between the two helical domains (residues 9-14) is appreciably different.

The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated, p-Cresolyl cobamide and 2-amino-vitamin B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were greater than or equal to 10(3) times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co-coordinated nucleotide. Pseudovitamin B12, with a weak Co-N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co-N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co-N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins. Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co-N coordinations, or the structures of their lower Co alpha ligands. Hence, the corrin ring, rather than a structural feature induced by the Co-N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.

The teichoic acid from the cell wall of Actinomadura cremea INA 292 has an unusual structure, being a poly(galactosylglycerol phosphate) chain with glycerol phosphate groups. Monomeric units of 1-O, beta-D-galactopyranosylglycerol monophosphate are joined in the polymer by phosphodiester links involving the glycerol C3 and the galactose C6 atoms. Approximately every second galactosyl substituent has a glycerol phosphate residue at its C3 atom. The teichoic acid structure was established by chemical analysis and 13C-NMR spectroscopy. There also is a peptidoglycan belonging to the A1 gamma type: as well as meso-2,6-diaminopimelic acid it contains small amounts of the LL form and glycine.

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be rapidly associated with the particulate fraction in a Ca(2+)-dependent manner at Ca2+ concentrations of 0.1-1.0 microM. This is also the range of the levels of intracellular Ca2+ reported for basal and various stimulated conditions. After translocation, phospholipase A2 could be released from the membranes in the presence of Ca2+ chelators, increasing the specific activity of phospholipase A2 in the supernatant fraction. These findings support the view that translocation is a regulatory mechanism of phospholipase A2 by bringing the enzyme to its substrate. Unlike the situation with protein kinase C, Mg2+ exerted little effect on phospholipase A2 translocation, indicating that this process is regulated in vivo mainly by fluctuations of the intracellular Ca2+ content.

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.

The tetracyclic polypeptide antibiotic cinnamycin (Ro 90-0198) belongs to the duramycin-type lantibiotics and contains the unusual amino acids threo-3-methyl-lanthionine, meso-lanthionine, lysinoalanine and 3-hydroxyaspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09-0198-producing strain Streptoverticillium griseoverticillatum via a 39-residue oligonucleotide probe derived from fragment 7-19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin-type prelantibiotics, this first duramycin-type prelantibiotic has an unusually long leader sequence of 58 amino acids. it also differs in the processing site and the direction of the formation of the threo-3-methyl-lanthionine bridges is from N-terminal cysteine to C-terminal dehydrated threonine residues, whereas the meso-lanthionine and lysinoalanine bridges are formed by addition reactions from C-terminal cysteine or lysine to N-terminal dehyrated serine residues.

We have isolated cDNAs encoding the alpha 6 subunit from a lambda gt11 expression library from human keratinocytes by combined screening with a rabbit polyclonal anti-alpha 6 antibody and the polymerase chain reaction. The alpha 6 subunit encoded by this cDNA consists of 1050 amino acids with a 991-amino-acid extracellular, a 23-amino-acid transmembrane and a 36-amino-acid cytoplasmic domain. The extracellular domain contains three putative divalent cation-binding sites and nine potential N-linked glycosylation sites. From a cDNA library from normal human mammary gland cells two different cDNAs for alpha 6 were isolated, one of which is identical to the above cDNA. The two alpha 6 subunits, called alpha 6A and alpha 6B, encoded by the two cDNAs each have a unique cytoplasmic domain, that of alpha 6B being 18 amino acids longer than that of alpha 6A. Different carcinoma cell lines contain transcripts for both alpha 6 subunits. K562 leukemic cells have little alpha 6A or alpha 6B mRNAs. The overall level of expression varies in the carcinoma cell lines, but reflects alpha 6 cell surface expression. In A375 melanoma cells, however, cell surface expression of alpha 6 was low in spite of a high level of mRNA. This suggest that other mechanisms may be involved in regulating the expression of alpha 6 on the surface of these cells. The mRNA for both alpha 6 subunits is around 6 kb. The alpha 6 subunits are similar to other alpha subunits (26-31% identity with cleaved alpha subunits) of the integrin family but they are more similar to the alpha 3 subunit (40% identity). This high degree of similarity may be the basis for their functional resemblance since both alpha 3 and alpha 6 subunits, when associated with beta 1, function as laminin receptors and bind to the long arm of laminin. The genes for alpha 6 and beta 4, the alternative beta subunit with which alpha 6 combines on certain epithelial cells, were mapped to chromosome 2 and 17q11-qter, respectively.

Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

In 87 randomly selected diabetic patients (67 type 1, 20 type 2) and 25 control subjects, gastric emptying of digestible solid and liquid meals and oesophageal transit of a solid bolus were measured with scintigraphic techniques. Gastrointestinal symptoms, autonomic nerve function and glycaemic control were evaluated in the diabetic patients. Gastric emptying and oesophageal transit were slower (P less than 0.001) in the diabetic patients compared with the control subjects, and each was delayed in about 40% of them. There was a relatively weak (r = 0.32; P less than 0.01) relationship between solid and liquid gastric emptying, and no significant correlation (r = 0.11, NS) between oesophageal transit and gastric emptying of the solid meal. Scores for upper gastrointestinal symptoms and autonomic nerve function correlated weakly (r = 0.21; P less than 0.05) with both oesophageal transit and gastric emptying. Gastric emptying of the liquid meal was slower (P less than 0.05) in patients with blood glucose concentrations greater than 15 mmol/l. These results indicate that gastric emptying in patients with diabetes mellitus should be assessed by liquid as well as by solid test meals and that oesophageal transit should not be used as a predictor of generalised diabetic gastroenteropathy.

Pneumocystis carinii pneumonia (PCP) has become a major cause of morbidity and mortality due to infectious diseases, largely as a result of the acquired immunodeficiency syndrome (AIDS) epidemic. Since the mortality from recurrent infection is between 40% and 60%, early diagnosis and therapy are the keys to survival. The role of technetium 99m diethylene triamine penta-acetate (DTPA) aerosol pulmonary clearance was studied in 81 patients with AIDS. The mathematical technique of curve stripping was found to be the optimal method of analysis and to provide an overall sensitivity of 94% for the detection of interstitial pneumonitis. The procedure was superior to standard pathology parameters and radiography and more convenient than gallium 67 scintigraphy.

Uptake of thallium 201 (201Tl) in the lungs has been proposed as a measure of left ventricular dysfunction with exercise. To study this hypothesis, we compared the lung/-heart (LH) ratio assessed from anterior planar images (ANT-P), from anterior images obtained during single photon emission tomography (SPET) acquisition (ANT-T) and from short-axis tomographic cross-sections (CS) in early post-exercise thallium 201 scintigrams. The study population consisted of 54 prepercutaneous transluminal coronary angioplasty (PTCA) studies (82% with single-vessel disease), 50 post-PTCA studies, 33 pre-coronary artery bypass surgery (CABG) studies (71% with three-vessel disease), 30 post-CABG studies and 30 patients with a left ventricular dysfunction (LVD) due to an acute myocardial infarction; 18 individuals with a low likelihood of coronary artery disease (CAD) served as a control group. The results demonstrated that, on average, the LH ratios obtained from ANT-P and ANT-T were not significantly different for all study groups; these ratios increased significantly with ischaemia and with LVD relative to non-ischaemic situations. However, the LH ratios in CS did not show a relation with ischaemia nor with LVD and differed significantly from the LH-ratios assessed from the anterior approaches. Each of the three approaches (ANT-P, ANT-T, CS) was characterized by large overlaps of LH ratios for the different study groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the reproducibility and response to change in end-tidal CO2 of a new method of quantifying regional mean cerebral transit time (MCTT) compared with the reproducibility and CO2 reactivity of middle cerebral artery (MCA) blood flow velocities measured using transcranial Doppler ultrasound. Within the range of end-tidal CO2 which could be achieved in conscious subjects breathing spontaneously, hemispheric MCTT, peak MCA velocity and mean MCA velocity showed a linear relationship with end-tidal CO2. After correction to a standardised end-tidal CO2, the coefficients of variation were 5.7% for hemispheric MCTT, 6.3% for peak MCA velocity and 6.8% for mean MCA velocity. Under the conditions of this study, MCA blood flow velocity was proportional to the reciprocal of MCTT, which in turn represents the ratio of blood flow to blood volume. Although the two methods appear to provide similar information, measurement of MCTT is quicker to perform, is less observer-dependent, provides regional information, uses conventional equipment present in most nuclear medicine departments and is less subject to problems associated with patient movement.

We used radiolabelled monoclonal antibodies (MoAbs) to prove disease persistence after treatment for ovarian carcinoma. Twelve patients with histologically confirmed ovarian carcinoma were studied. They received 5 mCi (1 mg) of iodine-131-B72.3 by intravenous injection before and after a complete course of chemotherapy. Images were obtained with a LFOV gamma camera 2 h after MoAb administration and daily up to 6 days. Before treatment 8 patients had a true positive scan. Questionable antibody uptake was observed in 2 patients while 1 had a true negative scan and 1 a false-negative examination. After treatment the therapeutic response was evaluated. Five patients had partial remission and antibody scan showed persistence of disease in all patients except 1. Four patients showed progression of the disease and 1 no change. The antibody scan was positive in 4 and questionable in 1. Two patients had complete remission and negative antibody scans. Computed tomography (CT) could not always discriminate postoperative fibrosis from tumour lesions, especially when the peritoneum was involved in the disease. High serum levels of tumour markers were constantly associated with the presence of tumour lesions, but normal values did not guarantee absence of disease. We conclude than the antibody scan is complementary to CT and serum tumor markers in the definition of therapeutic response.

Non-invasive quantification of renal function with radionuclides is an important role of nuclear medicine. With modern commercial preparations of technetium-99m diethylene triamine penta-acetic acid (DTPA), glomerular filtration rate (GFR) can be measured accurately either from the rate of disappearance of the tracer from plasma or from its rate of uptake into the kidneys. Determination of the latter with the gamma camera allows measurement of individual kidney GFR. Renal blood flow (RBF) can be measured from plasma clearance of hippurate or from first-pass kinetics of intravenously injected tracer using the gamma camera. The filtration fraction can be obtained from separate measurement of GFR and RBF, either globally from plasma clearance studies or of each kidney from gamma camera studies. Because they are central to the understanding of plasma clearance studies, the effective distribution volumes of the various tracers used for renal function studies are also discussed in detail.

This study investigated the conversations of 10 deaf children and their families at dinnertime and documented types of verbal exchanges, both spoken and signed, among family members. Results were compared with two other studies of deaf children's conversations--with teachers and with mothers during playtime. This study found that the deaf children responded more loquaciously to questions than they did to statements or expressions of ideas; and the children did not have success in continuing topics of conversation. Suggestions are presented to help families engage their deaf children in conversations in more depth.

This investigation was conducted to test the effectiveness of strategy teaching and sequencing practice problems in teaching students with learning disabilities to identify the correct algorithm for solving addition and subtraction word problems. Sixty-two students were assigned to one of three experimental groups: strategy plus sequence, strategy only, and sequence only. The results indicated that students in the strategy-plus-sequence group, as well as those in the strategy-only group, scored significantly higher than did students in the sequence-only group. Findings indicated that strategy teaching was the more effective of the two instructional components. Implications are discussed in terms of instructional design for students with learning disabilities.

This longitudinal study investigated the effects of time spent in academic instruction and time engaged on elementary students' academic achievement gains. Three groups were compared over grades as follows: (a) an at-risk experimental group of low-socioeconomic status (SES) students for whom teachers implemented classwide peer tutoring (CWPT) beginning with the second semester of first grade continuing through Grade 3; (b) an equivalent at-risk control group; and (c) a non-risk comparison group of students of average- to high-SES. In both the control and comparison groups, teachers employed conventional instructional practices over Grades 1 through 3. Results indicated significant group differences in the time spent in academic instruction, engagement, and gains on the subtests of the Metropolitan Achievement Test that favored the experimental and comparison groups over the control group. Implications include the effectiveness of CWPT for at-risk students and the continuing vulnerability of at-risk students whose daily instructional programs provide less instructional time and foster lower levels of active academic engagement.

High school youths who were prelingually and profoundly deaf, hearing elementary-school-age youths, and hearing reading-disabled high school youths read expository texts and filled in deleted words and phrases. After making word replacements, they explained their decisions in sign or verbally. As expected, the hearing youths had an easier time filling in the deleted portions and explaining what strategies they were using. While the deaf youths reported using similar strategies as the hearing youths, the frequency of each type of strategy differed. Deaf readers more often relied on rereading and background knowledge, while the hearing readers made greater use of context clues. The results suggest that instruction for deaf readers should include more effective comprehension strategies.

The BCL2 (B cell lymphoma/leukemia-2) proto-oncogene encodes a 26-kDa protein that has been localized to the inner mitochondrial membrane and that has been shown to enhance the survival of some types of hematopoietic cells. Here we show that NIH3T3 fibroblasts stably transfected with a BCL2 expression plasmid exhibit reduced dependence on competence-inducing growth factors (platelet-derived growth factor, PDGF; epidermal growth factor, EGF) for initiation of DNA synthesis. The importance of BCL2 for growth factor-induced proliferation of these cells was further confirmed by the useage of BCL2 antisense oligodeoxynucleotides. The mechanisms by which overexpression of p26 BCL2 contributes to fibroblast proliferation are unknown, but do not involve alterations in: (a) the production of inositol triphosphates (IP3), (b) PDGF-induced transient elevations in cytosolic Ca2+ ions, or (c) the activity of protein kinase C enzymes in these transfected cells. The results imply that changes in mitochondrial functions play an important role in the early stages of the cell cycle that render 3T3 cells competent to respond to the serum progression factors that stimulate entry into S-phase.

Cobalamin (Cbl, vitamin B12) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative EM radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. In contrast, in control cells, only 4.7% were assigned to lysosomes, with 47% to cytoplasm and 23.4% to mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.

The effects of microgravity on the immune system are largely unknown, but understanding such effects becomes increasingly important as space exploration continues and mission duration increases. Reductions in postflight human T cell reactivity to mitogens is well documented. Similar results have been obtained using a clinostat as an in vitro model of microgravity. In this study, a rat tail suspension model of weightlessness was used to examine in vitro lymphocyte proliferation in response to mitogens. Experiments were designed to uncover potential deficits in events related to proliferation including cell surface protein and IL-2 receptor (IL-2R) expression, interleukin-2 (IL-2) production, and accessory cells. Suspension of rats for 1 week led to a significant depression in [3H]thymidine incorporation by mitogen-stimulated peripheral blood lymphocytes (PBL) but only a small decrease in the proliferation of lymph node lymphocytes and splenocytes. There were no changes in the percentages of cells expressing CD4, CD5, CD8 or immunoglobulin. Moreover, no changes in IL-2 production or IL-2R expression were observed. More esterase-positive macrophages were detected in all lymphatic tissues of suspended rats, but there was no corresponding increase in the percentage of cells bearing the macrophage markers OX41 or OX42. This increase in the number of macrophages may be related to the observed suppression of lymphocyte proliferation. The tissue specificity of the decrease in mitogen activation indicates that there may be a compartmentalized response in the rats tested in the hindlimb suspension model.

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.

Skin fibroblasts from patients with inherited adenomatosis of the large bowel (ACR-SF) possess alterations in actin microfilament (MF) organization which serve to distinguish "predisposed" cells from fibroblasts derived from normal individuals (NSF). MF bundle frequency and diameter were considerably reduced in ACR-SF compared to NSF. This deficit in MF density correlated with a 60% decline in cytoskeletal-associated actin half-life. Absence of a well-structured MF network in ACR-SF was reflected in relatively poor cell-to-substrate adhesion (as indicated by increased sensitivity to trypsin release) and extensive membrane ruffling. Unlike NSF, ACR-SF failed to develop well-defined vinculin-containing focal contacts although the cellular content of vinculin was approximately the same in both cell types. The relatively low substrate adhesivity and reduced incidence of adhesive structures (i.e., MF and associated focal contacts) which typify ACR-SF correlated with a sixfold increase in cellular plasminogen activator (PA) activity. This increased protease activity corresponded with a 50-70% reduction in the content of the PA inhibitor-like protein p50 in both the saponin-resistant undersurface matrix and the culture medium. Increased motility and reduced cell-to-substrate adhesion, involving several cellular structural elements, appear to be significant correlates of the "predisposed" phenotype in cultured fibroblasts.

We have devised a technique that enables one to localize hyaluronate in cultured cells. Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence. The hyaluronate binding properties of this protein have been established. This technique was applied to the study of hyaluronate synthesis in glial cells. These cells do not themselves produce GHAP. O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats. These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium. In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced. The galC+ cells were invariably hyaluronate negative. Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells. The hyaluronate producing cells were typically small, process-bearing, and GFAP+. Some, but not all, were A2B5+ and could, therefore, be identified as type 2 (GFAP+, A2B5+) astrocytes. Type 1 (GFAP+, A2B5-) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate. Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes. This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.

Polyclonal IgG, prepared to a purified bovine cell surface sialoglycopeptide (SGP) inhibitor of cell division, was used to identify an antigenically related molecule on the surface of Swiss 3T3 cells. SDS-PAGE and Western analyses showed that the anti-SGP antibody was monospecific and primarily recognized a 66-kDA protein of 3T3 cell membranes. Treatment of intact 3T3 cells or 3T3 cell membranes with either broad and phosphatidylinositol-specific phospholipase C enzymes suggested that the antigenic material most likely existed as an integral membrane molecule, or associated as a multimeric complex, and was not anchored at the cell surface by a phospholipid. The addition of anti-SGP IgG to 3T3 cell monolayer cultures was shown to promote cell division, suggesting a regulatory function for the membrane-associated molecule.

We isolated 10 myoblast clones from Syrian/golden hamster embryo (SHE) cells irradiated with ultraviolet light. They were originally isolated as anchorage-independent clones. All clones showed characteristic morphology of myoblast in culture and formed swirled myofiber colonies specific to myoblast macrocolonies. However, in immunostaining experiments using anti-fast myosin antibody, we found that 4 of 10 myoblast clones were myosin negative (Myo-), while six were myosin positive (Myo+). Western blot analysis confirmed the disappearance of the 200-kDa myosin-specific band in the 4 Myo- clones. Furthermore, these 4 Myo- clones lost the ability to form multinucleated myotubes. Karyotype analysis revealed that all Myo- clones had trisomy of chromosome 7, while Myo+ clones showed no apparent karyotypic change from normal SHE cells. Compared with that of SHE cells, the transcriptional level of the myc gene in Myo+ clones was augmented, but there was no increase of myc gene expression in Myo- clones. Furthermore, the introduction of activated myc gene partially converted the Myo- phenotype to Myo+. These results suggest that trisomy of chromosome 7 and a deficiency in enhanced expression of the myc oncogene are associated with the suppression of both the production of myosin and the formation of multinucleated cells.

Tyrosine-specific protein kinase activity in neuronal differentiation was studied in a PC12 pheochromocytoma cell line (PC12-B9) produced by stable transfection with an inducible v-src gene encoding an activated tyrosine kinase (pp60v-src) under the transcriptional control of the mouse metallothionine I gene promoter. Induction of pp60v-src expression with Cd2+ and Zn2+ resulted in the reversible differentiation of PC12-B9 cells into neuron-like cells. pp60v-src elicited morphological differentiation with apparent first order kinetics at the same rate as NGF-directed neurite outgrowth in PC12-B9 cells. v-src gene expression enhanced the rate of NGF-directed neurite extension in an additive manner. Induction of pp60v-src alone constitutively increased the levels of phosphotyrosine-modified proteins (130-120, 90, 83, 65, 60/59, 36 kDa) detected by immunoblotting with phosphotyrosine antibodies. NGF treatment of PC12-B9 cells transiently increased the levels of distinct phosphotyrosine-modified proteins (108, 46, 42 kDa), as well as common substrates, including a 59-kDa protein that comigrated with alpha-tubulin. Phosphotyrosine-modified proteins were not synergistically increased in PC12-B9 cells induced for both v-src and NGF. The nonsynergistic effects of v-src gene expression on neurite outgrowth and phosphorylation suggest that pp60v-src induces PC12 cell differentiation by an intracellular signaling pathway that is largely distinct from that induced by NGF.

Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present.

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.

Differentiated mammalian cell lines can be isolated by immortalizing primary cells by transfection with DNA from plasmids containing sequences from SV40 early region. These cell lines show cytogenetic abnormalities but the degree of aneuploidy is considerably less than that observed in other established cell lines. No correlation was observed between the degree of differentiation of a clone and the extent of chromosomal damage.

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

Periosteal cells were enzymatically liberated from the tibiae of young chicks, introduced into cell culture, and allowed to reach confluence. The morphology of the cells gave the impression of a relatively homogeneous population of fibroblast-like cells. These cultured cells did not overtly express osteogenic or chondrogenic properties as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes. The cells were then replated at relatively high density and chronologically evaluated for the differentiation of bone and cartilage. These replated cells formed a multi-layer of fibroblast-like cells, the top portion of which eventually differentiated into bone tissue as evidenced by the presence of mineralization and immunocytochemical reactivity to bone Gla protein- and osteocyte-specific probes. Cells below this distinctive top layer differentiated into chondrocytes, which eventually further developed into hypertrophic chondrocytes as evidenced by their morphology and the presence of immunoreactive type X collagen in the matrix. Mineralization was also observed in the territorial matrix of these hypertrophic chondrocytes, when the culture was augmented with beta-glycerophosphate. Periosteal-derived cells replated at a lower density as controls did not show signs of osteochondrogenic differentiation. These observation suggest that periosteal-derived cells of young chicks contain mesenchymal cells which possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes depending on local environmental or positional cues.

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.

Cell spreading and cell locomotion arise from forces exerted by actin microfilaments upon the substratum. Using modified protein films at fluorocarbon oil--water interfaces as substrates, we have measured some minimal mechanical properties required of these films to support cell spreading forces in vitro. For murine 3T3-L1 fibroblasts, complete cell spreading was obtained when the films exceeded surface shear moduli and surface fracture points of 15 and 5 dyne/cm, respectively. The human WI-38 fibroblast required more robust films than did its transformed counterpart (WI-38/VA 13) in order to achieve equivalent spreading. These results are of significance in understanding the metastatic capabilities of cancer cells.

Lysine tRNA modification has been studied in mammalian ts-694 cells with respect to cell cycle progression in temperature downshift and upshift experiments. The modification of tRNA(lys) measured in temperature downshift experiments showed that tRNA(4lys) levels start to increase 6 h following the temperature shift, approximately 10-12 h prior to the cells entry into S phase. Ts-694 cells showed a gradual decrease in the level of tRNA(4lys) and the rates of DNA synthesis following a temperature upshift. The cells became growth arrested following incubation for 36-45 h at the rt. Cell cycle mapping of the temperature restriction point suggests a G1 block prior to the serum deprivation restriction point. Depletion of cellular tRNA(4lys) by serum deprivation followed by simultaneously shifting cells to the rt and feeding medium containing 10% serum showed that cells with low tRNA(4lys) levels and no mechanism for the synthesis of tRNA(4lys) could not enter S phase and synthesize DNA. Blocking of ts-694 at the G1/S boundary with aphidicolin indicates that cells that have passed through G1 are capable of entering S phase and synthesizing DNA independent of the incubation temperature. These results indicate that tRNA(4lys) is not needed during S phase for DNA replication but suggests that tRNA(4lys) is required for cells to progress through G1.

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.

Treatment of spontaneously differentiated PSMB embryonal carcinoma cells with murine interferon beta results in a transient decrease in the expression of the nuclear lamins A and C. Reduced levels of mRNAs were observed 4 h after the addition of interferon beta, with reductions in the polypeptides and assembled proteins within the nuclear lamina seen after 8 h of treatment. Expression of the 72-kDa (lamin A) and the 62-kDa (lamin C) polypeptides remained down regulated for 8 h, returning to control levels after 16 h of interferon treatment. The specificity of this response is indicated by the inhibitory action of a neutralizing antibody to interferon.

Mesenchymal cells from the wing buds of stage 24 chick embryos undergo differentiation to cartilage when plated at high density. Treatment of these cultures with phospholipase D resulted in inhibition of chondrogenesis. Phospholipase D treatment (which produces phosphatidic acid from membrane phospholipids) was found to affect cell proliferation and to dramatically increase intracellular free calcium levels and inositol phosphate production. Intracellular free Ca2+, mobilized as a result of phosphatidylinositol phosphate hydrolysis, may therefore inhibit chondrogenesis in embryonic mesenchymal cells.

Two-dimensional gel electrophoresis of cytosolic proteins from mature human erythrocytes combined with immunoblotting revealed the presence of a group of heat shock proteins (HSPs) that included two molecular chaperons of the HSP70 family (HSX70, inducible; HSC70, constitutively expressed) and HSP90. As expected for cells devoid of organelles, erythrocytes do not contain stress proteins that are localized either in the mitochondria (HSP60, glucose-regulated protein (GRP 75) or in the endoplasmic reticulum (GRP78 or Ig heavy chain-binding protein, endoplasmin). Since red cells are unable to replace proteins whose structure has been damaged by environmental changes the results are taken to imply a role for chaperons in monitoring, protecting, and maintaining the structure and stability of erythrocyte proteins.

After subcutaneous injections of doses of 5 x 2 and 1 x 10 mg cobalt (II) oxide/kg/week (total dose 1,000 mg/kg), respectively 5 out of 10 and 4 out of 10 rats in a lifetime study developed local malignant tumours (controls 0/10). After intraperitoneal injections of 3 x 200 mg cobalt (II) oxide/kg 14 out of 20 rats developed malignant intraperitoneal tumours (controls 1/20). With a Co/Al/Cr spinel the same treatment produced malignant intraperitoneal tumours in only 2 out of 20 rats. After intratracheal instillation of cobalt (II) oxide in single doses of 2 mg/kg (total dose 78 mg/kg) and 10 mg/kg (total dose 390 mg/kg) there were 2 benign pulmonary tumours among 100 rats in the low-dose group and 2 benign and 4 malignant pulmonary tumours among 100 rats in among 100 rats in the high-dose group. The Co/Al/Cr spinel was tested intratracheally only in the high dose (total dose 390 mg/kg); among the 100 rats this produced 3 malignant pulmonary tumours. Alternating intratracheal instillation of cobalt (II) oxide (total dose 470 mg/kg) and benzo[a]pyrene (total dose 200 mg/kg) led to 9 malignant pulmonary tumours in 20 rats, whereas benzo[a]pyrene alone caused only 1 malignant pulmonary tumour in 20 rats. The results suggest that cobalt (II) oxide (76.7% Co) has a weakly carcinogenic effect. The Co/Al/Cr spinel investigated is still less active, and even in very sensitive tests (i.p. and i.t. administration) shows no statistically significant carcinogenic effect. This smaller effect may possibly be explained by its lower cobalt content (24.0%) or its much lower solubility (only less than 10% of the solubility of CoO in 0.1 n HCl).

Sequential changes in rhodamine or fluorescein isothiocyanate-conjugated lectin binding of proximal and distal colonic crypts were studied during and after the administration of the 1,2 dimethylhydrazine (DMH). Five adult its unexposed to DMH or vehicle served as baseline controls. Tissue from normal appearing colon and tumor tissue was incubated with Ulex europaeus agglutinin 1 (UEA), Arachis hypogaea (PNA), Dolichos biflorus (DBA) and Griffonia simplicafolia 1 (GSA1). Distinct regional differences were noted in the baseline controls. UEA, PNA and DBA binding were absent in the distal colonic crypt cells. In the proximal colon minimal UEA and PNA binding was noted in the lower crypt whereas DBA binding was intense. GSA1 binding was diffuse in the upper and lower crypt of both regions. During carcinogenesis a progressive increase in PNA binding was noted in normal appearing colonic crypts from both regions. A progressive increase in PNA binding in proximal and distal colonic tumors was noted over time. Similar to normal tissue, DBA bound markedly to proximal colon tumors but was absent in most distal colonic tumors. UEA stained all proximal tumors intensely at all time points. In distal colonic tumors, UEA staining was diminished at 30 weeks compared to tumors analyzed at 16, 22 and 26 weeks. Mucin depletion was also a feature of tumor tissue compared to adjacent normal and hyperplastic glands. This study documents the region specific changes in lectin binding in normal and neoplastic colonic crypts induced by DMH.

Porcine pancreatic elastase (PPE) causes irreversible secretory cell metaplasia (SCM) in the intrapulmonary bronchi of hamsters. To determine whether this lesion can be induced in another rodent species, PPE was transorally instilled into the lungs of anesthetized rats. Saline-treated rats served as controls. Animals were killed 1 week, 3 weeks and 3 months after enzyme treatment and the lungs were inflation-fixed with 4% formalin/1% glutaraldehyde in 0.1 M Na cacodylate buffer at pH 7.4. Transverse sections from 3 intrapulmonary airway levels were embedded in paraffin and in glycol methacrylate. The secretory cell index (SCI) was determined from PAS-stained paraffin sections using a standardized scale of 0 to 4. There were no significant differences between SCI values of PPE-treated rats and saline-treated controls at any time point. Secretory cells in methacrylate sections were subclassified into S1, S2 and S3 cells on the basis of increasing amounts of PAS-positive intracellular granules. At one week, there was no effect of PPE on airway level I secretory cells, but significant increases in numbers of S2 and S3 cells were seen in level II and in S3 cells in level III. At 3 weeks, the number of S1 cells were increased in level I and S2 and S3 cells were increased in level III; level II secretory cells were unaffected. Level III secretory cells had returned to normal by 3 months. There were no significant differences between treatment groups in the total number of secretory cells (S1 + S2 + S3) at any airway level or time point examined.(ABSTRACT TRUNCATED AT 250 WORDS)

After in vitro-induced neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), altered actin organization in pulmonary epithelial cells was examined. A certain correlation was found between anchorage independency (AI), tumorigenicity in nude mice and altered actin organization. DNase I inhibition assay demonstrated significant loss of detergent-insoluble F-actin (stress fibers, p less than 0.02) and soluble F-actin (single filaments, p less than 0.0001) after AI transformation in comparison with untransformed cells, while the level of G-actin increased significantly (p less than 0.0001). At the same time, fluorescence and electron microscopy also revealed that after AI transformation there was a striking loss of stress fibers usually accompanied by reorganization of at least some of the lost stress fibers into F-actin aggregations. After s.c. implantation of AI-transformed cells into nude mice followed by recultivation of the developed tumors, DNase I-inhibition assay showed a significant increase (p less than 0.001) in the level of detergent-insoluble F-actin as compared with untransformed cells, but no significant difference in the amount of G-actin. However, most of this increased detergent-insoluble F-actin was in the form of aggregations as revealed by immunofluorescence and electron microscopy. This growth behaviour-dependent alteration in actin organization occurring after exposure to MNNG may be causally related to the progressive development of neoplastic phenotypes, although the biological significance of actin aggregation formation remains unclear. The results have also pointed out the importance of parallel investigations into both the biochemical and morphological statuses of actin, particularly when it may be regarded as an indicator of neoplastic transformation and malignancy.

Rats having received drinking water enriched with zinc (zinc acetate, 22.8 mmol/l) developed significantly more pulmonal metastases after an i.v. injection of 5 x 10(5) cultivated cells of a benzpyrene-induced sarcoma than receiving normal drinking water. Zinc ions seem to promote the emigration, implantation and outgrowth of circulating tumour cells.

Transplantation of pancreaticoduodenal allografts in the PVG.RT1c----PVG.RT1u high responder rat strain combination led to the evolution of haemorrhagic pancreatic necrosis over a rejection course of 8d. A cellular infiltrate consisting mainly of monocytes and macrophages was detectable in the stroma while acinar cells showed degranulation and a progressive decrease in number. From day 4 on apoptotic bodies could be identified ultrastructurally within the exocrine tissue. Apoptosis during pancreas rejection, previously undescribed, contributes to acinar cell loss after transplantation in this model.

One of the contributory causes to poor PRs in assisted reproduction has been the decreased viability of transferred embryos and the transfer of four-cell embryos into an environment that naturally would be receptive only to 5-day-old blastocysts. In this paper, we have reviewed our own work and that of others on the role of tubal ampullary cells (cocultures) to mimic the in vivo environment to bring about improved embryo quality and an increased number of blastocysts for replacement in IVF patients. The establishment, maintenance, and behavior of human tubal cell lines is first presented, followed by their use as cocultures for fertilization and cleavage of embryos. The mode of action, specificity, and cryopreservation of ampullary cells are also discussed. The currently available results of pregnancies after cocultures are presented together with future aspects of research that are necessary to refine the coculture system. The ultimate aim is to mimic in vivo conditions in vitro, so that at least the PRs of assisted conception can be parallel to normal fecundity in the human. Therefore, a very attractive future includes the freezing of blastocysts generated from coculture, thawing, and replacing them in natural cycles.

To evaluate the reliability of transvaginal ultrasound (US) and human chorionic gonadotropin (hCG) levels in detecting early abnormalities and predicting outcome of pregnancy.

One hundred thirty-two patients were studied, of which 113 had an intrauterine pregnancy and 19 had an ectopic pregnancy (EP).

In 78 with singleton normal pregnancies, US revealed a normal crown-rump length, heart motion, and hCG levels between 1,000 to 107,000 mIU/mL. Of 16 patients with small crown-rump length, heart motion present, and normal hCG levels, 6 aborted and 10 reached term. Thus, 6 of 84 (7.14%) singleton with fetal heart motion aborted. Thirteen with small crown-rump and absent heart motion also aborted. All 8 with an empty gestational sac aborted. In 8, transvaginal US detected four twins, two triplets, and two quadruplets, whereas hCG was not discriminative. Transvaginal US revealed an empty uterus in 19 patients with an EP, whereas serum hCG varied between 37 and 10,500 mIU/mL.

A fetal crown-rump length compatible with gestational age and fetal heart motion seen by transvaginal US can predict a term pregnancy in greater than 90% of patients.