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Biomedical subjects

G Stein

Publications and source records attributed to G Stein.

At least 19 recordsLinked to original sources

A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution.

The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes. The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis. The homologous mammalian ALR(Augmenter of Liver Regeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration. In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated. A temperature-sensitive mutant of the gene was stained with a mitochondria-specific dye and fluorescence was inspected at the permissive and restrictive temperature. A new phenotype for morphological defects of mitochondria was identified. Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell. After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed. Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell. GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol. Re-investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria.

Cell Polarity

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Animals

Lymphohematopoietic stem cell engraftment.

Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation. The stem cells that engraft in the nonmyeloablated are in G0, but are rapidly induced (by 12 hours) to enter the S phase after in vivo engraftment. Exposure of murine marrow to cytokines (IL-3, IL-6, IL-11 and steel factor) expands progenitor clones, induces stem cells into cell cycle, and causes a fluctuating engraftment phenotype tied to phase of cell cycle. These data indicate that the concepts of stem cell competition and fluctuation of stem cell phenotype with cell cycle transit should underlie any new stem cell engraftment strategy.

Animals

Investigations of bone turnover in renal osteopathy.

The renal bone disease which develops in chronic renal failure (CRF) is not an uniform disorder. Histomorphometry is accepted to be the best method for characterising the state of disease. The purpose of this study was to evaluate the suitability of pyridinium crosslinks in serum and urine as indicators of bone degradation processes. Patients with CRF had significantly higher Pyridinoline (Pyd) and Deoxypyridinoline (Dpyd) levels in serum and urine compared to normal controls except the urinary excretion in the subgroup of glomerulonephritis. A correlation was found between the serum levels of crosslinks and those of both creatinine and parathormone. The Pyd and Dpyd serum levels in patients under dialysis treatment were significantly higher than those of normal controls. With regard to bone turnover urinary crosslink measurements are of minor importance in CRF. In contrast, serum measurements could be helpful in revealing bone resorption both in patients with CRF and those under dialysis treatment.

Adult

Chemotyping of yeast mutants using robotics.

By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

Antifungal Agents

Polyplex-mediated gene transfer into human retinal pigment epithelial cells in vitro.

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age-related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome-mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex-mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex-mediated gene transfer, but stable integration does occur at low frequencies with and without selection.

DNA

Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA fragmentation.

Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very useful preparation. Enterocytes towards the apex of the villi become increasingly susceptible to apoptosis. Here, this "apoptotic gradient" is used to demonstrate the presence of BAX and BCL-2 proteins in the cytoplasm of cells at the onset of apoptosis. In semithin serial sections of the small intestine, BAX, BCL-2 and DNA fragmentation were demonstrated. BAX and BCL-2 are always colocalized and only in cells with fragmented DNA. The gradient of BAX or BCL-2 staining is similar to the gradient of DNA fragmentation. Immunoreactivity for BCL-2 or BAX is most intense in cells that are prone to become apoptotic next in the course of cellular turnover but not in cells in an advanced apoptotic state, showing strongly condensed chromatin. When using the same technique on semithin sections of kidney biopsies, containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Similar to the situation in the enterocytes, BAX staining was confined to the cytoplasm of epithelial cells with a moderate degree of DNA fragmentation and reduced in epithelial cells with a high degree of DNA fragmentation. In contrast to the situation in the small intestine, very low levels of BCL-2 were found. The results suggest that expression of BCL-2 and BAX is related to cell damage as indicated by DNA fragmentation but not to advanced stages of cellular death, as indicated by chromatin condensation and cellular shrinkage.

Animals

Outcome in refractory depression.

BACKGROUND: Failure to respond to first-line antidepressant treatment can occur in up to 40% of patients with depressive illness. A proven strategy for managing this refractory depression is lithium augmentation. The long-term outcome and optimal management of patients treated with lithium augmentation remains unclear. We describe a 4-8 year naturalistic follow-up of patients treated with lithium augmentation in two controlled studies of its efficacy in refractory depression. METHOD: Cases were followed up with personal interview where possible, and by telephone and general practitioner contact otherwise. Lifetime clinical status was ascertained using the Schedule for Affective Disorders and Schizophrenia-Lifetime (SADS-L). RESULTS: We obtained outcome data on 53 of the original eligible 76 patients. There was a good outcome in 38 (72%) patients. Good outcome was associated with a less endogenous nature of depression and an absence of previous hospitalisations. CONCLUSIONS: There do not seem to be any specific prognostic indicators of long-term outcome to lithium augmentation beyond those recognised to be relevant in the outcome of depression generally. LIMITATIONS: The conclusions are limited by incomplete follow-up of the total original sample and lack of objective illness and medication data for the intervening period.

Adult

Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization.

The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.

Amino Acid Sequence

The influence of ofloxacin (Tarivid) on the parasite-host inter-relationship in patients with chronic urinary tract infection.

In urinary tract infection (UTI), the aim of antimicrobial chemotherapy is either to attenuate the virulence of infective micro-organisms, to influence the interaction between germs and host or to kill bacteria. In 25 females (aged 49.8+/-14.7 years) with chronic UTI, parameters of inflammation (CRP, alpha 2-globulin, leukocytes, ESR) as well as renal function were analyzed under the treatment with ofloxacin 2 x 200 mg. Subsequently, bacterial attachment, bacterial count, leukocytes, antibody-coated bacteria (ACB), immunoglobulin (Ig) G, albumin, a2-microglobulin, Tamm Horsfall protein, secretory IgA (sIgA) and lysozyme were determined in urine. The micro-organisms were examined with regard to the expression of hemolysin, aerobactin, P-fimbriae and according to their plasmid profile. Ofloxacin serum levels were analyzed once prior to, on day 6 during and on day 3 after drug administration. In all cases, the acute clinical symptoms had disappeared after 10 days of treatment, all bacteria were eliminated, and the parameters of inflammation in serum and urine had returned to normal. On the sixth day of therapy, no expression of P-fimbriae was detectable in the Escherichia coli strains isolated, and the attachment rate decreased from 42+/-30.9% to 11.1+/-18.1%. The sIgA level rose from 42.6+68.5 prior to therapy to 88.8+/-136.8 mmol/l on day 3 after therapy; acute symptoms of UTI did not recur in any case during the period of 1 year.

Adult

Cell cultures from cryopreserved renal biopsies and other tissue samples.

Answering questions regarding research or clinical aspects, histological and histochemical examinations of tissue specimens are playing an increasing role. The same is true for cell cultures obtained from organ specimens. In most cases, tissue samples are obtained only once and have to be examined immediately. This is often impracticable and therefore, it is necessary to store tissue samples or cells from established cell cultures so as to be able to continue examinations at a later time. Very rare reports exist on the preservation of tissue for performing cell culture examinations and they exclusively refer to tumour tissue and bone marrow, but not to normal organ tissue and biopsy samples. Therefore, in this study cell cultures from several organs have been prepared immediately after obtaining the tissue and compared with those established after cryopreservation several months later. The tissue specimens were obtained from rats (kidney, skin, heart) and from humans (kidney, placenta) or were biopsy specimens from the kidney and skin. From all these cryopreparations, typical cells were cultured. There was no significant difference in the mean population doubling time (MPD) and regarding morphological cell criteria between cell cultures obtained from fresh tissue samples after biopsy and those prepared several months after cryopreservation. There was nearly the same ratio between most cell types present in the tissue. From these results we can conclude that in the examined organs cells from cryopreserved tissue can be cultured even months or more than one year later. At least, these results make it possible to answer new questions and repeat different experiments at any time.

Actins

Physiological doses of calcium regulatory hormones do not normalize bone cells in uraemic rats.

BACKGROUND: Low bone turnover despite normal parathyroid hormone (PTH) concentrations has been found in many patients with end-stage renal failure. Hyporesponsiveness to the calcaemic action is also a known feature of uraemia. Hyporesponsiveness of bone surface cells involved in bone modelling has not been demonstrated to date. It was the purpose of this study using a rat model of moderate renal failure to investigate whether doses of PTH and calcitriol that reverse the effect of parathyroidectomy on calcaemia also normalize bone surface cell activity. MATERIALS AND METHODS: Sham-operated pair-fed male Spraque-Dawley rats were compared with subtotally nephrectomized (SNX), parathyroidectomized (PTX) rats that received either solvent or calcitriol (5 pmol kg -1 h-1) + 1,34 rat PTH (100 ng kg -1 h-1) by osmotic mini-pump. Histomorphometric measurements were carried out in the vertebral body (L5). RESULTS: In SNX/PTX animals, calcitriol + 1,34 rat PTH caused a modest increase in serum calcium (S-Ca) within the normal range. Osteoclast surface per cent was significantly lower in solvent-treated SNX/PTX rats than in sham-operated controls [3.7 +/- 2.8 osteoclast surface/bone surface (OcS/BS%) vs. 6.3 +/- 3.9], and this was not normalized by PTH + calcitriol (3.3 +/- 3). In contrast, osteoblast surface per cent and osteoid surface per cent were increased over values in sham-operated rats; as a result, co-administration of calcitriol and 1,34 rat PTH caused a highly significant increase in fractional bone volume (BV/TV). CONCLUSIONS: The results show that administration of PTH and calcitriol in doses that raise serum calcium fails to normalize the percentage of osteoclast surface, but was effective in raising osteoblast number and osteoblast volume in experimental renal failure. The results argue for abnormal response of bone cells to calcium-regulating hormones and/or the action of factors other than calcium regulatory hormones in the genesis of skeletal abnormalities of renal failure.

Animals

[Serum level-adjusted dosage of once-daily aminoglycoside therapy in critical illness: results of a prospective study].

OBJECTIVE: In critically ill patients, the adjustment of target peak and trough levels of tobramycin was investigated because aminoglycoside pharmacokinetics can be changed by multiple influences. Sufficient but not too high peak serum concentrations and low trough levels, however, should be achieved to ensure a therapeutic effect and to minimize toxicity. METHODS: 70 critically ill patients of 51 +/- 18 years were monitored daily during their aminoglycoside treatment on the intensive care unit targeting a peak of about 12 micrograms/ml 30 minutes after infusion and a trough level below 1 to 2 micrograms/ml. Dose recommendations were given daily, taking into consideration serum levels, dose predictions (Bayesian method, ABBOTTBASE), creatinine clearance and clinical findings. Creatinine clearance was estimated according to the Cockcroft-Gault-formula as well as directly by the urine collection method. RESULTS: The standardized initial dose of 400 mg tobramycin led to average peak serum levels of 14.2 +/- 3.9 micrograms/ml in the patients with an apparent distribution volume of 0.345 +/- 0.074 L/kg. In 95% of the patients, the initial peak was higher than 8.5 micrograms/ml; levels higher than 20 micrograms/ml were observed in 7%, extremely low concentrations (below 5 micrograms/ml) in 2%. With individually adjusted doses between 160 and 560 mg, a mean peak of 11.5 +/- 2.7 micrograms/ml was measured subsequently. The levels amounted to 96 +/- 23% of the predicted values, deviations greater than 50% occurred in 5%. The target trough level was achieved in 99%, in less than 3% the dosing interval was extended up to 72 hours. A tobramycin clearance below 80 ml/min/1.73 m2 was associated with average 80% and 33% higher creatinine clearance values according to the Cockcroft-method and the direct method, respectively. CONCLUSION: Target peak and trough aminoglycoside levels are adjustable even in critically ill patients. Reduced tobramycin clearance can be associated with normal creatinine clearance. Assuming an exact methodology, a reduced "direct" creatinine clearance, however, indicates a reduced drug clearance.

Anti-Bacterial Agents

Correlates between hematopoiesis and neuropoiesis: neural stem cells.

There are many parallels between the neuropoietic and lymphohematopoietic systems. The lymphohematopoietic stem/progenitor cell system has been extensively characterized, but there are still major questions relating to the definitive stem cell assay, the structure of the system (i.e., hierarchical versus cell cycle-based), and the nature of differentiation (i.e., stochastic versus deterministic). Recent data have established the existence of an epidermal growth factor (EGF)-responsive neural stem cell in adult mice. We have studied these neural progenitor/stem cells in fetal (day 15) and 2-day postnatal mice and established a single-cell progenitor assay and a variety of putative uni-, bi-, and tripotential stem cells that form in response to EGF. Neurospheres are the EGF-responsive neural units that grow in liquid culture, and we have found that cells derived from these neurospheres express a wide array of cytokines and their receptors. This will provide a window on the hemopoietic progenitor system analogous to that created by the description of in vitro growth of clonal hematopoietic progenitors.

Animals