Tubular absorption of filtered cystatin-C in the rat kidney

As shown in previous studies, the two basic proteins aprotinin (Ap, 6.5 kDa) and cystatin (Cy, 13.3 kDa) can be used to estimate whole kidney glomerular filtration rate by measuring the renal cortical uptake relative to plasma concentration after i.v. injection. Local uptake of Ap can also be used to estimate local filtration rate, and the present experiments were undertaken to examine whether Cy would give a similar uptake pattern. Ap and Cy were labelled with 131I and 125I, respectively, and injected as an i.v. bolus. Frequent blood samples provided information on the filtered load. Five to 20 min after injection the kidney was clamped, frozen, and five tissue samples of 5-10 mg each were cut out from outer (OC), middle (MC) and inner cortex (IC) to be weighed and assessed for radioactivity. Five minute clearance ratios, Cy:Ap, were 1.36 +/- 0.04, 1.27 +/- 0.03 and 1.19 +/- 0.04 in OC, MC and IC, respectively. The higher Cy clearance was expected from a higher glomerular filtrate:plasma ratio of the less basic Cy (Donnan distribution). However, this does not explain the increase of Cy:Ap clearances going from IC to the OC. A surplus of extracellular uptake of Cy in superficial layers was excluded, leading to the following interpretation. In all cortical layers the proximal convoluted tubule, i.e. the protein uptake segment, is located more superficially than its parent glomerulus. A longer uptake segment for Cy than for Ap will therefore lead to a relative greater transfer of filtered Cy from IC to MC, and from MC to OC. Anatomical studies on single nephrons presented in another article lend strong support to this interpretation.     

Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format

A system for microarrays was developed to detect and differentiate Cucumber mosaic virus (CMV) serogroups and subgroups. The coat protein genes of 14 different isolates were amplified using cy3-labelled generic but species-specific primers. These amplicons were hybridized against a set of five different serotype and subgroup specific 24-mer oligonucleotides bound to an aldehyde-coated glass slide via an aminolinker. The results of the hybridization revealed that the method allowed a clear differentiation of the 14 different CMV isolates into the serogroupes 1 and 2, and in addition was able to assign 9 out of 10 different serogroup 1 isolates correctly into subgroups 1a and 1b. This differentiation was not possible by RFLP analysis with the restriction enzyme MspI. The use of amplicons larger than 700 base pairs and their successful differentiation by hybridization to specific oligonucleotides opens avenues to highly parallel, yet sensitive assays for plant viruses.     

Regulation of hepatocyte activator inhibitor-1 expression by androgen and oncogenic transformation in the prostate

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347-355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.     

Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD₄₅₀) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD₄₅₀ turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD₄₅₀ turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.     

Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes

Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.     

F2-isoprostanes stimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis?

Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F2-isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl4-induced hepatic fibrosis, plasma F2-isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F2-isoprostanes in the concentration range found in the in vivo studies (10(-9)-10(-8) M), a striking increase in DNA synthesis (reversed by the thromboxane A2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F2-isoprostanes markedly increased the production of transforming growth factor-beta1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion

Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells.     

Allele variations in the OCA2 gene (pink-eyed-dilution locus) are associated with genetic susceptibility to melanoma

The occuloalbinism 2 (OCA2) gene, localized at 15q11, encodes a melanosomal transmembrane protein that is involved in the most common form of human occulo-cutaneous albinism, a human genetic disorder characterized by fair pigmentation and susceptibility to skin cancer. We wondered whether allele variations at this locus could influence susceptibility to malignant melanoma (MM). In all, 10 intragenic single-nucleotide polymorphisms (SNPs) were genotyped in 113 patients with melanomas and in 105 Caucasian control subjects with no personal or family history of skin cancer. By comparing allelic distribution between cases and controls, we show that MM and OCA2 are associated (p value=0.030 after correction for multiple testing). Then, a recently developed strategy, the 'combination test' enabled us to show that a combination formed by two SNPs was most strongly associated to MM, suggesting a possible interaction between intragenic SNPs. In addition, the role of OCA2 on MM risk was also detected using a logistic model taking into account the presence of variants of the melanocortin 1 receptor gene (MC1R, a key pigmentation gene) and all pigmentation characteristics as melanoma risk factors. Our data demonstrate that a second pigmentation gene, in addition to MC1R, is involved in genetic susceptibility to melanoma.     

Autonomie in der Offenen Kinder- und Jugendarbeit fördern – Ergebnisse eines partizipativen Evaluationsprojekts

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz - Partizipation zu ermöglichen ist Teil des Auftrags der Offenen Kinder- und Jugendarbeit. Dazu gehört die Einbindung der...