bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunochemical analysis of the Kx protein from human red cells of different Kell phenotypes using antibodies raised against synthetic peptides
Authors: CARBONNET FLORENCE , HATTAB CLAUDE , COLLEC EMMANUEL , LE VAN KIM CAROLINE , CARTRON JEAN‐PIERRE , BERTRAND OLIVIER
Published: November 2003

The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription‐translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell–Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including K0, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in K0 cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.
Interferon-gamma (IFN-γ) down-regulates the rhinovirus-induced expression of intercellular adhesion molecule-1 (ICAM-1) on human airway epithelial cells
Authors: SETHI S K , BIANCO A , ALLEN J T , KNIGHT R A , SPITERI M A
Published: November 2003

Human rhinoviruses (HRV) are a major cause of upper respiratory tract infections in man, and can exacerbate existing pulmonary disease. The major group of HRV attach to ICAM-1, which is expressed on nasal and bronchial epithelial cells. To study the influence of biological mediators on ICAM-1 expression, and consequently HRV attachment and infection, we compared the effects of various cytokines, alone and in combination, on ICAM-1 expression by an uninfected and HRV-infected bronchial epithelial cell line H292. Cytokines known to be released soon after viral infection, such as tumour necrosis factor-alpha (TNF-α), IL-1β and the chemokine IL-8 increase ICAM-1 expression on uninfected cells. Epithelial cells infected with live HRV-14 displayed marked up-regulation of ICAM-1 compared with baseline. TNF-α further enhanced the HRV-induced increase in ICAM-1 expression on epithelial cells, peaking at day 4 after infection, whilst IL-8 exhibited a steady increase in ICAM-1 expression over 14 days. In contrast, IFN-γ, a known Th1 antiviral lymphokine, whilst increasing the level of ICAM-1 on uninfected cells, induced a significant persistent down-regulation of ICAM-1 expression on HRV-infected epithelial cells. With combinations of TNF-α and IFN-γ, ICAM-1 expression on HRV-infected cells was reduced to basal levels. The effects of IFN-γ were paralleled by a reduction in viral titres. Our in vitro model has provided useful insights into the early pathogenic events of HRV infection at the level of the host cell–virus interaction. Our data confirm that biological mediators play a crucial role in the pathogenesis as well as the course of HRV infection which is modulated by the types, and time kinetics of inflammatory cytokines in the immediate microenvironment.
Humoral immune response within the lung in HIV-1 infection
Authors: MOJA P H , JALIL A , QUESNEL A , PEROL M , COTTE L , LIVROZET J M , BOIBIEUX A , CHAMSON A , VERGNON J M , LUCHT F , TRAN R , POZZETTO B , GENIN C
Published: November 2003

In order to study the humoral immune defences in the respiratory tract during HIV-1 infection, we measured the levels, local productions and anti-HIV and antibacterial activities of IgG and IgA in the bronchoalveolar lavage fluid (BALF) and serum of 61 adult patients with severe HIV infection and of 56 HIV− controls. Albumin was used as the serum transudation factor. The increase of immunoglobulin levels in the serum of HIV-infected patients was confirmed. The IgG level was also increased in epithelial lining fluid (ELF), whereas the total IgA level was unchanged and secretory IgA (SIgA) level was decreased. The ELF/serum immunoglobulin ratios suggested that the IgG present in ELF resulted mainly from transudation, in contrast to SIgA, which was synthesized locally in controls but greatly diminished in HIV-infected patients. IgG to HIV-1 could be detected in BALF of all the patients, but IgA to HIV-1 only in 30% of patients. BAL IgG reacted more consistently and with a broader array of HIV-1 antigens than did IgA. BAL IgA, when present in samples, reacted primarily with viral envelope antigens. Because IgA specificities to some HIV-1 antigens were detected more intensively by BAL than by serum immunoglobulins, we conclude that the mucosal immune response is distinct from that in serum. IgG antibody activity to Streptococcus pneumoniae was decreased in HIV-infected patients' sera, and IgA antibody activities to S. pneumoniae and to Pseudomonas aeruginosa were decreased in ELF in HIV-infected patients.
Infection with rat cytomegalovirus (CMV) in the immunocompromised host is associated with the appearance of a T cell population with reduced CD8 and T cell receptor (TCR) expression
Authors: VAN DAM J G , DAMOISEAUX J G M C , VAN DER HEIJDEN H A M D , GRAULS G , VAN BREDA VRIESMAN P J C , BRUGGEMAN C A
Published: November 2003

Infection with human cytomegalovirus (HCMV) mostly results in a chronic subclinical infection; the immune system is unable to eliminate the virus and is apparently in equilibrium with the persistent virus. In the immunosuppressed host this equilibrium is disturbed, resulting in clinical infection. Rat cytomegalovirus (RCMV) infection in its host can be used as a model for HCMV infection. Using flow cytometry we examined the effect of acute RCMV infection on the composition of leucocyte subsets in the peripheral blood of both immunocompetent and immunosuppressed (5 Gy total body irradiation) Lewis rats. Special attention was paid to the natural killer (NK) cells and the CD8+ T cells known to be involved in the control of viral infections. Furthermore, we determined the presence of leucocyte subsets in the internal organs by immunohistochemistry. In immunocompetent rats, infection caused a small increase in NK cells and a large increase in CD8+ T cells. In contrast, infection of immunosuppressed rats caused a marked increase in NK cells and a small increase in CD8+ T cells, consisting of T cells with reduced expression of both CD8 and TCR. This phenomenon is characteristic of anergic CD8+ T cells, possibly explaining the ability of the virus to escape elimination by the immune system. The increase of NK cells in the peripheral blood of immunosuppressed, RCMV-infected rats could also be detected in kidney, liver, lung and pancreas, but not in salivary gland. This could explain the long persistence of infectious virus in the salivary gland.
Amino-terminal identity of co-existent amyloid and non-amyloid immunoglobulin κ light chain deposits. A human disease to study alterations of protein conformation
Authors: KAPLAN B , VIDAL R , KUMAR A , GHISO J , FRANGIONE B , GALLO G
Published: November 2003

Tissue deposition of monoclonal immunoglobulin light chains is a serious complication in some patients with B cell proliferative disorders. The deposits are typically fibrillar and Congophilic in amyloid (AL) and non-fibrillar and Congophobic in light chain deposition disease (LCDD), and rarely coexist in the same patient. From post-mortem tissue of an individual with fibrillar and non-fibrillar κ light chain deposits in different sites, we separately extracted and analysed biochemically and immunochemically the non-amyloid deposits from isolated glomeruli, the amyloid from isolated renal arteries and the amyloid from myocardium in which the only deposits were amyloid restricted to mural arteries. Western blotting analysis of both the extracted amyloid and the non-amyloid deposits demonstrated 25-kD bands immunoreactive with anti-κ antibody, and the identity of the N-terminal amino acid sequences that belong to the variable region κIV light chain subgroup. This is the first human disease in which antigenically similar but morphologically different deposits have been separately biochemically analysed. We propose that combined LCDD and AL is an ideal human disease to study the relationships and the factors that influence the conversion of non-amyloidogenic to amyloidogenic conformations.
Impaired Ca2+ mobilization by X-linked agammaglobulinaemia (XLA) B cells in response to ligation of the B cell receptor (BCR)
Authors: GENEVIER H C , CALLARD R E
Published: November 2003

XLA bone marrow samples were shown to contain B cells expressing IgM, and pre-B cells that express the μ-surrogate light chain (μψLC) complex, albeit at a reduced frequency to that found in normal bone marrow. Antibody ligation of μ heavy chain on these cells and an XLA B cell line did not induce a Ca2+ flux, whereas ligation of μ heavy chain on normal bone marrow cells, μψLC+ pre-B cell lines and an IgM+ B cell line did. The block in XLA B cells was not due to a defect in the basic mechanism of Ca2+ flux generation, as the cells responded well to thapsigargin. In addition, the defect did not affect T cells, which were shown to respond to CD3 antibody with a Ca2+ flux. Ligation of μ heavy chain on XLA bone marrow cells did, however, activate tyrosine kinases, resulting in tyrosine phosphorylation of a cellular protein with a molecular weight of approximately 115 kD. These results indicate that Btk may be necessary for the generation of the Ca2+ flux in response to ligation of μ heavy chain on B cells and μψLC+ pre-B.
T cell receptor (TCR) BV gene repertoires and clonal expansions of CD4 cells in patients with HIV infections
Authors: Roglic M , Macphee R D , Duncan S R , Sattler F R , Theofilopoulos A N
Published: November 2003

Despite extensive investigation, the pathogenesis of T cell depletions that characterize AIDS has not been elucidated. To study this process further, we evaluated T cell antigen receptor β-chain variable gene (TCRBV) repertoires in peripheral blood lymphocytes (PBL) of 23 HIV-infected patients. Expression levels of 28 TCRBV were determined by multiprobe RNase protection assay after polymerase chain reaction (PCR) amplifications. Abnormal expansions (>2 s.d. from mean normal values) were frequent in HIV CD4, accounting for 26% of total measured TCRBV in this population. The number and magnitude of abnormalities among individuals were inversely proportional to their CD4 counts (P<0.012 and P<0.01, respectively). While abnormalities were not randomly distributed among TCRBV subfamilies, no particular genes were expanded or contracted among all patients. Only 14% of CD8 TCRBV were proportionally expanded (P<0.01 compared with CD4), and there were limited concordances between paired CD8 and CD4 repertoires among individuals. CDR3 length analyses and TCRBV sequencing showed that most CD4 expansions comprised clonal or oligoclonal populations. Thus, T cell responses in HIV patients are characterized by severe TCRBV biases and clonal expansions among CD4 subsets, and these processes are exaggerated with disease progression. The heterogeneity and oligoclonality of the TCRBV expansions are consistent with responses to HIV-encoded or other conventional antigens rather than superantigenic effects. The presence of CD4 clonal proliferations in these patients may be important in the pathogenesis of HIV, and the absence or reduction of many T cell specificities due to oligoclonal expansions may increase susceptibility to opportunistic infections.
Febrile infection changes the expression of IgG Fc receptors and complement receptors in human neutrophils in vivo
Authors: Leino L , Sorvajärvi K , Katajisto J , Laine M , Lilius E-M , Pelliniemi T-T , Rajamäki A , Silvoniemi P , Nikoskelainen J
Published: November 2003

We have examined the expression of the IgG Fc receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3) in neutrophils from 42 patients with febrile bacterial infection, 20 patients with febrile viral infection and 69 non-febrile healthy individuals. Using receptor-specific MoAbs and immunofluorescence flow cytometry the relative fluorescence intensity (as a measure of receptor number) and the proportion of receptor-positive cells were determined in peripheral blood neutrophils exposed to minimal processing, consisting only of erythrolysis. Both the percentage of FcRI-positive neutrophils and FcRI number per neutrophil were significantly (P<0.001 and P<0.0001) increased in bacterial infected patients compared with controls, whereas in viral infected patients only the FcRI percentage was markedly elevated (P<0.05). In addition, both FcRII and CR1 levels were significantly higher in the bacterial infection group than in the viral infection and control groups (bacterial versus control P<0.001, bacterial versus viral P<0.0001). No changes in expression of FcRIII or CR3 were found in the patient groups. The kinetic analysis of receptor expression in bacterial infection patients revealed a shift in the percentage of FcRI-bearing neutrophils towards normal values already on day 2 after the first analysis. On the other hand, the levels of FcRI, FcRII and CR1 remained clearly elevated in these patients during 1 week's follow-up period. We conclude that febrile infection may cause systemic activation of the entire pool of circulating neutrophils, resulting in alterations in cell surface receptor expression, some of which are characteristic of the nature of the infectious agent.
Growth hormone responses to growth hormone‐releasing hormone and clonidine in patients with type I diabetes and in normal controls: effect of age, body mass index and sex
Authors: Jacobs M. L. , Nathoe H. M. W. , Blankestijn P. J. , Stijnen Th. , Weber R. F. A.
Published: November 2003

OBJECTIVE Increased plasma concentrations of GH and increased GH responses to provocative stimuli are reported in patients with poorly controlled type I diabetes and are suggested to be related to complications. Our aim was to investigate GH concentrations in moderately controlled patients.

PATIENTS AND MEASUREMENTS We have investigated IGF‐I concentrations and fasting GH concentrations and the response to 1 μg/kg body weight GH‐releasing hormone (GHRH) intravenously and/or to 150 μg clonidine intravenously in 77 moderately controlled patients with type I diabetes and in 46 healthy controls.

RESULTS Median HbA1c in the patients was 8.5% (upper level of normal 6.3%). Fasting GH and GH concentrations after the administration of GHRH were not significantly different in patients with type I diabetes compared with normal controls. Fasting and stimulated GH concentrations after the administration of clonidine were significantly higher in the patients, but this could be explained by their lower age and body mass index compared with controls. In controls but not in patients there was a negative correlation between GH and glucose concentrations. IGF‐I was significantly lower in patients with diabetes than in controls, even after correction for age, body mass index and sex.

CONCLUSIONS Patients with moderately controlled type I diabetes mellitus have normal baseline and stimulated GH concentrations after the administration of GHRH or clonidine compared with healthy controls, when corrected for age, body mass index and sex. However, these ‘normal’ GH concentrations must be considered inappropriately high in view of the hyperglycaemia in these patients. The low plasma IGF‐I concentrations might be responsible for the GH over‐production.
The GnRH challenge test: a quantitative measure of pituitary desensitization during GnRH agonist administration
Authors: Scheele Fedde , Hompes Peter G. A. , Lambalk Cornelis B. , Schoute Erik , Broekmans Frank J. , Schoemaker Joop
Published: November 2003

OBJECTIVE The use of GnRH agonists for desensitization of the pituitary is widespread in gynaecological practice. For indications such as contraception and treatment of uterine leiomyomata partial desensitization may suffice. With respect to partial desensitization of the pituitary we have addressed three basic questions: (1) Is the degree of pituitary desensitization dependent on the dose of agonist used? (2) What is the optimal way to measure the degree of pituitary desensitization? (3) Is it possible to create a standard to express the degree of pituitary desensitization?

DESIGN AND PATIENTS Twenty‐four women were randomized into 4 groups of 6 women. To achieve pituitary desensitization, the groups received 0.1, 0.25, 0.5 and 1.0 μg/min GnRH respectively, for 6 weeks.

MEASUREMENTS Pituitary desensitization was measured by gonadotrophin levels and responses to a 100‐μg bolus of GnRH and an oestradiol benzoate challenge‐test.

RESULTS The level of LH and the responses of LH and FSH to the GnRH challenge showed significant dose‐dependent suppression. Multiple regression indicated the LH response to the GnRH challenge was the best way to measure pituitary desensitization. From the LH responses to the GnRH‐challenge a ‘standard curve’ was established for the assessment of the degree of pituitary desensitization.

CONCLUSION The LH response to the GnRH challenge test is the best available measure of pituitary desensitization during GnRH agonist treatment.
The effects of different sampling techniques on smear quality and the diagnosis of cytological abnormalities in cervical screening
Authors: WILLIAMSON S. L. H. , HAIR T. , WADEHRA V.
Published: November 2003

A major cause of false‐negative cervical smears is sampling error. We examined the results obtained with three different instruments in 126 608 smears from general practitioners. The spatula/brush combination yielded the highest proportion of smears showing cytological abnormalities, and the Cervex brush the lowest. Although not a randomized study, this paper highlights the shortcomings of the Cervex brush. We postulate a mechanical deficiency. Diagnostic accuracy rather than a high proportion of good quality smears should dictate the choice of instrument.
The effect of metoprolol treatment on insulin sensitivity and diurnal plasma hormone levels in hypertensive subjects
Authors: Gudbjörnsdóttir S. , Fowelin J. , Elam M. , Attvall S. , Bengtsson B.‐Å. , Mårin P. , Lönnroth P.
Published: November 2003

To evaluate the effect of metoprolol on insulin sensitivity and diurnal plasma hormone levels, seven mildly hypertensive subjects were investigated (four men and three women, age 52 ± 8, body mass index 25.4 ± 1.9, mean ± SD). The study had a placebo‐controlled, double‐blind, crossover design with 6 weeks’ metoprolol treatment (100 mg b.i.d) vs. placebo. At the end of each treatment period 24‐h blood samples were collected continuously for diurnal analysis of hormone levels and a hyperinsulinaemic euglycaemic clamp combined with [3‐3H]‐d‐glucose infusion was performed. Insulin sensitivity was evaluated by means of three different methods: diurnal plasma insulin and glucose levels; glucose consumption; and insulin sensitivity index during euglycaemic clamp conditions. Fasting blood glucose and insulin concentrations as well as mean plasma diurnal levels of insulin, growth hormone, testosterone and cortisol were similar after placebo and metoprolol treatment, whereas noradrenaline and adrenaline levels were significantly increased after metoprolol. During the clamp, plasma insulin was significantly higher after metoprolol treatment than after placebo treatment (56 ± 3 vs. 64 ±2 mU L–1, P < 0.05). Consequently, the insulin sensitivity index [glucose infusion rate (GIR)/plasma insulin] was lower after metoprolol treatment (16.1 ± 2.6 vs. 10.2 ± 1.2, P < 0.05), although GIR was not significantly changed. We suggest that the insulin sensitivity index may not accurately reflect the insulin effect as the plasma level of insulin was significantly increased during insulin infusion but not at 24 h, possibly because of alteration of distribution and/or degradation rate of exogenous insulin. Thus, the likelihood of metoprolol inducing insulin resistance in hypertensive subjects may be less than previously proposed.
Capillary filtration coefficient and urinary albumin leak at altitude
Authors: LEWIS D. M. , BRADWELL A. R. , SHORE A. C. , BEAMAN M. , TOOKE J. E.
Published: November 2003

Rapid ascent to altitude risks the development of acute mountain sickness. This study demonstrates changes in peripheral capillary filtration coefficient and renal protein loss in subjects suffering from various degrees of mountain sickness after passive ascent to 4559 m. Capillary filtration coefficient of the calf capillary bed, measured by computer‐based multistep strain gauge plethysmography, increased significantly after 23.5 h at altitude when symptoms were most severe: 4.45 (2.76–6.03) to 6.31 (3.86–11.07) ml min–1 per 100 g of tissue mmHg–1, median (range) (P <0.02). Urinary albumin excretion was increased after one night at altitude from 1.1 (0.6–1.5) to 2.45 (1.0–6.8) mg of albumin per mmol of creatinine (P <0.05). These results demonstrate simultaneous leakage of a peripheral capillary bed to fluid measured by strain gauge plethysmography, and renal albumin leak, and suggest a systemic process of increased capillary leakage for different‐sized molecules caused by rapid exposure to hypobaric hypoxia.
Plasma catecholamine concentrations in essential hyperhidrosis and effects of thoracoscopic D2–D3 sympathicolysis
Authors: NOPPEN M. , SEVENS C. , GERLO E. , VINCKEN W.
Published: November 2003

Essential hyperhidrosis (EH) is caused by a poorly understood overactivity of the sympathetic fibres passing through the upper dorsal sympathetic ganglia D2 and D3. These ganglia are also in the pathway of the sympathetic innervation of the heart and lungs. Therefore, although the predominant sympathetic neurotransmitter at the eccrine sweat glands is acetylcholine, the plasma concentration of noradrenaline (NA) (which is the main sympathetic neurotransmitter at the end organs including the heart and the lungs) may be elevated. Furthermore, as there are some indications for generalized sympathetic overactivity in EH, the plasma concentration of adrenaline (A) may also be elevated. Plasma levels of NA and A were therefore determined in 13 EH patients before and after thoracoscopic D2–D3 sympathicolysis (TS). Preoperative NA and A plasma levels were all within the normal limits used in our laboratory. After TS, mean NA plasma levels are significantly decreased, whereas mean A are unchanged. We conclude that sympathetic overactivity in EH is limited to the upper dorsal sympathetic ganglia and that some of the cardiovascular and pulmonary effects that are observed after TS may be associated with the decrease in NA.
Serum leptin concentration and fuel homeostasis in healthy man
Authors: Tuominen J. A. , Ebeling P. , LaQuier F. W. , Heiman M. L. , Stephens T. , Koivisto V. A.
Published: November 2003

We studied the interrelationship between the obese gene product serum leptin, insulin and counterregulatory hormone concentrations and glycogen synthesis in 26 healthy men. A 4‐h euglycaemic insulin clamp with muscle biopsies was performed after a resting control day in 26 subjects, and in 14 of them also after heavy, glycogen‐depleting (32%, P < 0.01) exercise. Serum leptin concentrations were at baseline 34% (0.67  ±  0.18 vs. 1.03  ±  0.13 ng L−1, P  ±  0.05) lower after the exercise, and rose during hyperinsulinaemia by 56% (to 1.38  ±  0.19 ng L−1, P < 0.001) and 34% (to 1.05 ± 0.20 ng L–1, P < 0.01) after the post‐exercise and control studies respectively. Basal serum leptin concentration correlated positively with body mass index (r = 0.42, P < 0.05), serum cortisol concentration (r = 0.70, P < 0.001) and the rise in muscle glycogen content during the clamp (r = 0.43, P < 0.05) and inversely with serum growth hormone concentration (r = −0.43, P < 0.05). There was a positive correlation between serum leptin after hyperinsulinaemia and serum insulin concentration during the hyperinsulinaemia (r = 0.42, P < 0.05). After exercise, basal serum leptin level correlated with serum triglyceride concentration (r = 0.82, P < 0.001) and after hyperinsulinaemia serum leptin correlated positively with muscle glycogen content (r = 0.56, P < 0.05). It was concluded that serum leptin concentrations correlate directly with serum insulin, cortisol and triglyceride and inversely with growth hormone concentrations. They are decreased by glycogen‐depleting exercise and increase during hyperinsulinaemic clamp. These data suggest that leptin is associated with factors regulating fuel homeostasis and its hormonal control in man.
Emission and content of monoterpenes in intact and wounded needles of the Mediterranean Pine, Pinus pinea
Authors: Loreto F. , Nascetti P. , Graverini A. , Mannozzi M.
Published: November 2003

1. Biogenic emission of isoprenoids may be involved in protecting plants against pathogens and may interfere with the chemistry of atmosphere. Pinus pinea (L.) is one of the most widespread conifers in the Mediterranean area and is a potentially strong emitter of monoterpenes.

2. We demonstrated by gas‐exchange studies that monoterpenes other than trans‐β‐ocimene are not emitted significantly by intact primary and secondary needles of 3‐year‐old P. pinea plants.

3. Trans‐β‐ocimene emission is light‐ and CO2‐dependent. Therefore this compound is probably formed from photosynthetic carbon in the chloroplasts and does not originate from the same biochemical route as other monoterpenes stored in resin ducts.

4. Trans‐β‐ocimene emission starts in spring, peaks in summer and ceases at the end of summer. This strong seasonality may mark a phase‐shift in needle ontogeny, but is not related strictly to changes in photosynthesis.

5. Wounded needles, either illuminated or darkened, massively release monoterpenes contained in the ducts, primarily limonene and α‐pinene. Monoterpene release from wounded needles rapidly decreases but does not cease altogether, even after 24 h. The amount of monoterpenes emitted from wounded needles in 24 h can be estimated at about 1200 and 120 µmol g −1 day−1 (dry mass) of limonene and α‐pinene, respectively. Wounded needles are a main contributor to monoterpene emission by vegetation.

6. Monoterpene content is two to three times greater in primary than in secondary needles. In primary needles, typical only of young plants, the elevated content of monoterpenes may allow plant survival against pathogenic attacks.
Diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli
Authors: Nishimura Akiko , Moriya Shigeki , Ukai Hideki , Nagai Kazuo , Wachi Masaaki , Yamada Yuko
Published: November 2003

Background:

The timing of the cell division in Escherichia coli is highly regulated, but its mechanism has not been identified. Previously we have found that the cfcA1 mutation uncouples DNA replication and cell division and elevates the frequency of cell division. We further analysed the structure and the role of the cfc genes of cfcA11, a derivative of cfcA1, and another cfc mutant, cfcB1.

Results:

The cfc mutants divided prior to the ordinary stage of cell division and produced many small cells with nucleoid. However, the cells grew exponentially, and the length of a cell cycle and the initiation mass for chromosome replication were not altered by the cfc mutations. These properties resulted from a reduction of the period between the nucleoid division and the cell division in a cell cycle, and a compensatory lengthening of the period between the cell division and the initiation of the next round of DNA replication. CfcA11 has a mutation in glySa which encodes the α‐subunit of glycyl‐tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH+, and the intracellular level of Ap4A in cfcA1 was 15‐fold higher, and cfcB1 was 100‐fold higher than their parent. Experiments using wild‐type cells showed that a high level of Ap4A caused early cell division, and that a low level of Ap4A caused delayed cell division.

Conclusion:

Ap4A is a signal for the induction of cell division. High levels of Ap4A are responsible for the initiation of cell division. The glyS mutation permits an efficient synthesis of Ap4A.
Sendai virus‐based expression of HIV‐1 gp120: reinforcement by the V(−) version
Authors: Yu Deshan , Shioda Tatsuo , Kato Atsushi , Hasan Mohammad K. , Sakai Yuko , Nagai Yoshiyuki
Published: November 2003

Background:

We have established a system for recovering Sendai virus (SeV), a nonsegmented negative strand RNA virus, entirely from cDNA at an extremely high rate, and have succeeded in creating a V(−) SeV whose gene expression was greatly enhanced by the deletion of the nonessential V gene. Because of its extreme medical importance, there has been a strong need for the establishment of a better system to express the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV‐1) in sufficient quantity and purity. It also remains to be established to produce gp120 in in vitro natural host cells for HIV‐1 such as human primary blood mononuclear cells, macrophages or established T cell lines.

Results:

Using the above system, we created recombinant Sendai viruses expressing the gp120 in CV1 cells, a monkey kidney line. The expression level from the standard V(+) version has already reached 2.2 μg per 106 infected cells, which was readily purified from the culture fluid with a recovery rate of about 60%, and has so far appeared to be functionally and serologically authentic. The inserted gp120 gene was stably maintained during numerous passages of the recombinant virus. The V(−) version‐based expression was even more robust, consistently reaching over 6.0 μg per 106 cells, a level that is one of the highest currently attainable for gp120 production in mammalian cells. Furthermore, a broad host range of SeV allowed gp120 production in all the three natural host cells for HIV‐1 described above.

Conclusions:

SeV‐based expression serves as a novel choice for producing large quantities of HIV‐1 gp120 and will greatly facilitate biochemical, biological and immunological studies of this important glycoprotein.
Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell‐cycle dependent expression
Authors: Yokoi Masayuki , Ito Masaki , Izumi Masako , Miyazawa Hiroshi , Nakai Hirokazu , Hanaoka Fumio
Published: November 2003

Background:

DNA polymerase α has been studied in considerable detail in yeast and animals. Genetic and biochemical analyses reveal that this enzyme is composed of a heterotetramer and is necessary for replicon initiation and primer synthesis in lagging strand synthesis. In spite of the fact that modes of DNA replication in plants seem to be similar to those in other eukaryotes, very little is known about the biochemical components that participate in DNA replication of plants, including DNA polymerases.

Results:

Using a 561‐base pair DNA fragment, obtained by polymerase chain reaction amplification from a rice cDNA library as a probe, we isolated and sequenced a cDNA homologous to the cDNA for the catalytic subunit of rice DNA polymerase α. The encoded polypeptide has extensive homology with the catalytic subunit of DNA polymerase α from several species. Furthermore, when the cDNA was expressed in eukaryotic transcription/translation systems, the protein products showed DNA polymerase activity which was inhibited by a monoclonal antibody specific for DNA polymerase α. Using RNA gel blot analysis, we found that the levels of mRNA of the catalytic subunit of this enzyme is regulated during the cell‐cycle in plant cells.

Conclusion:

This is the first report which describes the cDNA cloning of plant DNA polymerase. We conclude that the principal features of the DNA polymerase α catalytic subunit are conserved in plants.
Combined laparoscopic and vaginal surgery: saving of time by means of a new type of OR draping cloth
Authors: Brandner P. , Neis K. J.
Published: November 2003

Objective

To describe our experience with a new operating room drape designed specifically for laparoscopic surgery where changes between the abdominal and the vaginal route are frequent.

Design

The drape was tested in the 373 laparoscopic operations including 32 laparoscopically assisted vaginal hysterectomies (LAVHs) carried out over a 4‐month period. Special interest was given to possible saving of time.

Results

The amount of time saved depended on the number of changes between the vaginal and the abdominal sites, which varies according to the type of operation performed. In LAVH, the pre‐ and intraoperative preparation time was reduced by almost 50%.

Conclusion

Since the drape has been proved to shorten operating time, it may be a benefit to the patient as well as to surgeons and their staff.
Immunodetection of androgen receptor in human urinary bladder cancer
Authors: ZHUANG Y.‐H. , BLÄUER M. , TAMMELA T. , TUOHIMAA P.
Published: November 2003

We investigated the expression of androgen receptor (AR) protein in transitional cell carcinoma of human urinary bladder in paraffin‐embedded sections of tumours obtained from nine patients with urinary bladder cancer treated by radical cystectomy. In addition, immunoblotting of AR was also performed on selected samples. Nuclear immunoreactivity of AR was found in seven of the nine urinary bladder cancers studied. AR showed variable staining intensity within a tumour. In the immunoblots, a 110 kDa AR signal was seen with anti‐AR antibody, and faint bands of 90 and 60 kDa were also observed. Immunohistochemistry of p53 and c‐erbB‐2 was also carried out and compared with the distribution of AR. The high frequency of AR expression suggests a role for androgens in transitional cell carcinoma of human urinary bladder.
Meningeal melanocytoma: case report and review of the literature
Authors: IBÁÑEZ J. , WEIL B. , AYALA A. , JIMENEZ A. , ACEDO C. , RODRIGO I.
Published: November 2003

We report a case of meningeal melanocytoma in the thoracic spinal cord of a 44‐year‐old woman and review previously documented cases. Our patient experienced numbness and tingling in her left leg for 8 years, and low back pains with intermittent claudication for the previous 2 months. A histologically benign 20‐mm tumour was totally resected. Radiation therapy was not given. The tumour showed the histological, immunohistochemical and ultrastructural features of a meningeal melanocytoma. The patient is alive without recurrence 4.5 years after surgery.
Interferon‐γ differentially regulates antigen‐processing functions in distinct endocytic compartments of macrophages with constitutive expression of class II major histocompatibility complex molecules
Authors: HOCKETT R. D. , COOK J. R. , FINDLAY K. , HARDING C. V.
Published: November 2003

RAW264.7 cells were transfected to express constitutively the murine class II major histocompatibility complex (MHC‐II) molecule, I‐Ak. The resulting RAW.Ak cells presented HEL(46–61) peptide to 3A9 T hybridoma cells, but they were unable to process and present HEL protein in their resting state. However, IFN‐γ stimulation induced the ability of RAW.Ak to process and present HEL protein, with little effect on their ability to present HEL(46–61) peptide. Antigen catabolism showed little change with IFN‐γ stimulation, suggesting that the production of peptides was not the regulated step in the processing pathway. Furthermore, HEL(46–61) peptide delivered directly into lysosomes by acid‐resistant liposomes was also presented only upon IFN‐γ stimulation, while the presentation of peptides delivered into endosomes by acid‐sensitive liposomes showed a lesser dependence on IFN‐γ stimulation. Thus, IFN‐γ regulated the ability of peptides delivered into certain lysosomal compartments to meet with MHC‐II molecules and form peptide–MHC complexes, or to transport subsequently to the plasma membrane. Two other antigens, ribonuclease A and haemoglobin, were processed by RAW.Ak cells without IFN‐γ stimulation, suggesting that these antigens could be processed by different mechanisms, perhaps in earlier endocytic compartments. Thus, different antigens may be processed in distinct endocytic compartments, and an IFN‐γ‐regulated mechanism controls the rescue of peptides from lysosomal compartments for presentation at the plasma membrane.
Expression of β2‐microglobulin‐free HLA class I α‐chains on activated T cells requires internalization of HLA class I heterodimers
Authors: PICKL W. F. , HOLTER W. , STÖCKL J. , MAJDIC O. , KNAPP W.
Published: November 2003

HLA class I molecules on activated T cells are expressed as heterodimers associated with β2‐microglobulin (β2‐m) and also β2‐m‐free HLA class I α‐chains. Mechanisms leading to the expression of the activation associated β2‐m‐free HLA class I α‐chains are poorly defined, however. Upon enzymatical removal of HLA class I α‐chains on activated T cells, re‐expression is observed within minutes upon reculture, reaching half‐maximal levels within 1 hr. This process is independent of de novo protein synthesis and of export of newly synthesized proteins. Inhibition of the formation of coated pits by potassium depletion of cells abrogated the re‐expression of HLA class I α‐chains, suggesting that recycling events of HLA class I heterodimers via endosomal compartments are required for the generation of monoclonal antibody LA45‐reactive α‐chains. Furthermore, the rate of α‐chain generation seems to be governed by the amount of cell surface‐expressed HLA class I heterodimers. Taken together these findings suggest that β2‐m‐free HLA class I α‐chains are generated during the process of class I heterodimer recycling.
Characterization of a mast cell line that lacks the extracellular domain of membrane c‐kit
Authors: MEKORI Y. A. , OH C. K. , DASTYCH J. , GOFF J. P. , ADACHI S. , BIANCHINE P. J. , WOROBEC A. , SEMERE T. , PIERCE J. H. , METCALFE D. D.
Published: November 2003

Expression of the c‐kit proto‐oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin‐3 (IL‐3)‐dependent mast cell line CFTL‐15 lacks the extracellular domain of the c‐kit receptor. This observation was made after noting that the c‐kit ligand stem cell factor (SCF) could not prevent IL‐3 deprivation‐induced mast cell apoptosis and that CFTL‐15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti‐c‐kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c‐kit on CFTL‐15 cells. There was no identifiable message for the extracellular domain of c‐kit in these cells, as determined by reverse transcriptase‐polymerse chain reaction (RT‐PCR). These previously unrecognized properties of the CFTL‐15 mast cell line allowed the examination of other biological consequences of the lack of c‐kit on mast cells. Analysing the ability of these cells to adhere to surface‐bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL‐15 mast cells did not adhere to fibroblasts, which is known to require c‐kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL‐15 cells underwent apoptosis on removal of IL‐3 coincident with a decrease in endogenous Bcl‐2 mRNA. Overexpression of Bcl‐2 cDNA prolonged survival of Bcl‐2‐transfected CFTL‐15 cells upon withdrawal of IL‐3. Thus, the CFTL‐15 cell line that lacks surface c‐kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c‐kit and provide a novel experimental model for further investigation.
C2‐ceramide and C6‐ceramide inhibited priming for enhanced release of superoxide in monocytes, but had no effect on the killing of leukaemic cells by monocytes
Authors: NAKABO Y. , PABST M. J.
Published: November 2003

Ceramide acts as an intracellular second messenger in cellular signal transduction. We examined the effects of two cell‐permeable ceramides, C2‐ceramide and C6‐ceramide, on human monocyte functions. After monocytes were primed with lipopolysaccharide (LPS) or interferon‐γ (IFN‐γ) for 18 hr in suspension culture, they produced a high amount of superoxide (O−2) when triggered by phorbol myristate acetate. C2‐ or C6‐ceramide inhibited O−2 release from monocytes primed with LPS (1 ng/ml) or IFN‐γ (100 U/ml), but did not affect unprimed monocytes. An analogue, C2‐dihydroceramide, was inactive. C2‐ceramide was most effective at 6 μm, and C6‐ceramide at 60 μm. C2‐ or C6‐ceramide at these concentrations was not toxic for monocytes, as assessed by trypan blue exclusion and by the 3‐[
4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT) assay which measures the ability of live cells to produce formazan. C2‐ceramide (20 μm) had no effect on the killing of leukaemic cells (HL‐60 and K562 cells) by monocytes treated with IFN‐γ, LPS, or both for 18 hr, with killing assessed by an 111Indium‐releasing assay. C2‐ceramide (20 μm) induced secretion of low amounts of tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) from the monocytes. But C2‐ceramide did not alter the higher secretion of TNF‐α or IL‐1β from monocytes treated with IFN‐γ or LPS. Thus, the cell‐permeable ceramides acted like antagonists of LPS, rather than analogues of LPS, as has been proposed. The results here showed that the signal transduction pathway for O−2 release by monocytes differed from that for the cytolysis of leukaemic cells, and confirmed that oxygen radicals are not involved in cytolysis.
Rapid method for resectioning of light microscopy sections for electron microscopy
Authors: Kristóf Z.
Published: November 2003

A rapid method is described for obtaining ultrathin sections from light microscopy sections. Five‐micrometre epoxy sections, heat‐flattened to slides, were affixed to the tips of plastic blocks by light‐curable dental bond, and cured while still on the microscope stage by illumination with blue light for 2 min. Sections were detached from the slides by rapid cooling and then resectioned for electron microscopy.
The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three‐dimensional reconstruction
Authors: Beniac D. R. , Czarnota G. J. , Rutherford B. L. , Ottensmeyer F. P. , Harauz G.
Published: November 2003

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion‐assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three‐dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441–444, 1995; Stark et al., Structure, 3, 815–821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.
Transmission electron microscopy applied to the study of works of art: sample preparation methodology and possible techniques
Authors: SAN ANDRES M. , BAEZ M. I. , BALDONEDO J. L. , BARBA C.
Published: November 2003

Technical examination of a work of art is a necessary preliminary stage both for proper conservation/restoration of the work and for purposes of dating and/or authentication. There is a wide variety of methods and procedures, and of these a particularly valuable technique is stratigraphic analysis in view of the data that it furnishes on the composition of the pictorial layers of which a painting is composed. The techniques utilized in this type of analysis to date have been essentially light microscopy and scanning electron microscopy. Transmission electron microscopy can provide new data for characterization of pictorial layers, thanks to the possibility of individually using ultrathin sections of paint sample. This study provides morphological analysis and microanalysis by X‐ray energy dispersion, with determination of the crystalline structure of each particle by electron diffraction. The sample preparation method for producing thin sections from the pictorial layers for examination in the TEM is described. This allows the stratigraphic section to be preserved exactly as applied by the artist. The first results from the examination of three microsamples from actual old works of art are presented. The individual components of each strata were successfully identified in all cases.
Directional analysis of digitized planar sets by configuration counts
Authors: JENSEN E. B. V. , KIDERLEN M.
Published: November 2003

The oriented rose of normal directions of a random set Z can be used to quantify its anisotropy. A method for estimating this quantity from a digitization of Z in a sampling window, i.e. its pixel image, is presented. The image is analysed locally by considering pixel squares of size n × n. This allows us to count the number of different types of n × n configurations in the pixel image. We show that it suffices to restrict attention to the so‐called informative configurations. The number of informative configurations increases only polynomially in n. An algorithm to find these informative configurations is presented. Furthermore, estimators of the oriented rose based on counts of observed informative configurations are derived. The procedure is illustrated by a simulated example and an analysis of a microscopic image of steel.
Quantitative microstructure analysis of polymer‐modified mortars
Authors: Jenni A. , Herwegh M. , Zurbriggen R. , Aberle T. , Holzer L.
Published: November 2003

Digital light, fluorescence and electron microscopy in combination with wavelength‐dispersive spectroscopy were used to visualize individual polymers, air voids, cement phases and filler minerals in a polymer‐modified cementitious tile adhesive. In order to investigate the evolution and processes involved in formation of the mortar microstructure, quantifications of the phase distribution in the mortar were performed including phase‐specific imaging and digital image analysis. The required sample preparation techniques and imaging related topics are discussed. As a form of case study, the different techniques were applied to obtain a quantitative characterization of a specific mortar mixture. The results indicate that the mortar fractionates during different stages ranging from the early fresh mortar until the final hardened mortar stage. This induces process‐dependent enrichments of the phases at specific locations in the mortar. The approach presented provides important information for a comprehensive understanding of the functionality of polymer‐modified mortars.