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Biomedical subjects

G Wolf

Publications and source records attributed to G Wolf.

At least 217 records · Page 12Linked to original sources
PubMed1998

Potassium-stimulated taurine release and nitric oxide synthase activity during quinolinic acid lesion of the rat striatum.

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K+-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K+-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K+-induced taurine release. We conclude that a substantial part of K+-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K+-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.

Animals↗
PubMed1998

Preimplantation diagnosis of thalassemias.

PURPOSE: Preimplantation genetic diagnosis (PGD) is an important option for couples at risk of having children with beta-globin mutations to avoid selective abortions of affected fetuses following prenatal diagnosis. METHODS: We performed PGD for thalassemia in 12 clinical cycles (IVS1-110, and IVS-745 mutations) using biopsy of the first and second polar bodies (PBs) extruded from oocytes during maturation and fertilization, coupled with nested polymerase chain reaction analysis and restriction digestion. RESULTS: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resulted in the selection and transfer of 30 unaffected embryos (2.5 embryos per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly polymorphic linked markers, a short tandem repeat immediately at the 5' end of the globin gene and HUMTH01 which is a syntenic short tandem repeat. The application of multiplex polymerase chain reaction of the beta-globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes. CONCLUSIONS: Confirmation studies of the embryos resulting from the oocytes predicted to contain an affected gene confirmed the diagnosis in 98% of the cases, thus demonstrating the accuracy and reliability of PB PGD of thalassemia mutations. The application of PB analysis in six patients resulted in two ongoing pregnancies with a thalassemia-free fetus already confirmed in both of them by prenatal diagnosis.

Alleles↗
PubMed1998

Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis.

PURPOSE: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. METHODS: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. RESULTS: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15 pregnancies are ongoing after being confirmed normal by prenatal diagnosis. CONCLUSIONS: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.

Aneuploidy↗
PubMed1998

Allelic loss on chromosome 10q in human lung cancer: association with tumour progression and metastatic phenotype.

We analysed 78 carcinomas of the lung for allelic losses on chromosome 10q. The tumours were of different stage and grade and comprised 22 small-cell lung carcinomas (SCLC), 40 squamous cell carcinomas (SCC), 11 adenocarcinomas, four large-cell carcinomas and one carcinoid. They were investigated by six polymorphic markers located between 10q21 and 10qter. We observed a high incidence of loss of heterozygosity (LOH) in SCLC (91%) and metastatic SCC (56%). Non-metastatic SCC showed deletions in three cases (14%) and no LOH was found in the other types of non-small-cell lung cancer. The statistical analysis indicated that the presence of LOH correlated significantly with advanced tumour stages in the entire collective and in particular within the SCLC and SCC subgroups. For SCC, a positive association was found between LOH and metastases formation, while in SCLC the number of non-metastatic tumours was too small for a final conclusion. Whereas SCLC was frequently characterized by multiple allelic losses, suggesting the deletion of the entire chromosomal arm, SCC showed interstitial imbalances. A high incidence of allelic loss was observed between the markers D10S677 and D10S1223. The analysis of five informative cases suggested the presence of two non-overlapping regions between the loci D10S677/D10S1237 and D10S1213/D10S1223. In SCLC, we did not find mutations in the putative tumour-suppressor gene MXI1. The data indicate that LOH on chromosome 10q is associated with tumour progression in SCC and SCLC. Thus it may become a useful genetic marker in the assessment of the malignant potential of these tumour types.

Alleles↗
PubMed1998

Correlation between binding and dynamics at SH2 domain interfaces.

Protein recognition is a key determinant in regulating biological processes. Structures of complexes of interacting proteins provide significant insights into the mechanism of specific recognition. However, studies performed by modifying residues within a protein interface demonstrate that binding is not fully explained by these static pictures. Thus, structural data alone was not predictive of affinities in binding studies of phospholipase Cgamma1 and Syp phosphatase SH2 domains with phosphopeptides. NMR relaxation experiments probing dynamics of methyl groups of these complexes indicate a correlation between binding energy and restriction of motion at the interfacial region responsible for specific binding.

Insulin Receptor Substrate Proteins↗
PubMed1998

Gastrin/cholecystokinin type B receptors in the kidney: molecular, pharmacological, functional characterization, and localization.

BACKGROUND: Gastrin/cholecystokinin type B receptors (CCKBRs) can be found on parietal cells and smooth muscle cells and are the predominant brain CCK receptors. Recent cloning studies indicate that this is receptor type might also be expressed in the kidney. MATERIALS AND METHODS: We used Northern blot analysis in guinea pig. kidney and reverse transcriptase polymerase chain reaction (RT-PCR) in several murine kidney cells lines to evaluate this organ for the expression of the CCKBRs. The receptor was pharmacologically characterized by displacement experiments using [125I]-BH-CCKs and various agonists and antagonists. Polyclonal antibodies vs. the CCKBRs were raised in chicken, and immunohistochemistry on tissue sections was used to localize the receptor within the organ. The effect of gastrin on renal cell growth was measured using proximal tubulus (MCT) cells, which were cultured with gastrin (10-9 M) for 24-72 h. Cell counts and [3H]-thymidine incorporation experiments were performed. RESULTS: CCKBR transcripts can be detected in kidney RNA (tubules > glomeruli > interstitium). RT-PCR revealed CCKBR transcripts in proximal tubules (MCT cells) and in mesangium (MMC). The medullary thick ascending limb of Henle's loop and several control tissues such as liver and muscle were negative. Displacement experiments using [125I]-BH-CCK and various agonists and antagonists identified binding sites with typical CCKBR pharmacology. CCKBRs were localized in the proximal tubulus, distal collecting ducts and mesangium cells. Treatment of rested MCT cells with gastrin 17-1 induced cell proliferation and [3H]-thymidine incorporation by at least 40% compared with normal growth (P < 0.05). CONCLUSION: These results show for the first time that CCKBRs are present in selected areas of the kidney, and strongly confirm our previous observation that this organ expresses binding sites for [125I]-gastrin. Furthermore, gastrin might act as a growth factor in the kidney.

Animals↗
PubMed1998

Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals.

BACKGROUND: Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular cells. We have previously demonstrated that this Ang II-mediated hypertrophy occurs in the G1-phase of the cell cycle and depends on the induction of p27Kip1, an inhibitor of G1-phase cyclin/cyclin-dependent kinase complexes. The present study was undertaken to investigate whether Ang II may stimulate superoxide anions (O2.) formation in cultured LLC-PK1 and cultured mouse proximal tubule (MCT) cells, and to gain further insight into a potential relationship between O2. and cell cycle regulation. METHODS: Reactive oxygen species were measured with the lucigenin method in intact cells. The effects of various inhibitors were tested on Ang II-induced O2. production. Cells were transiently transfected with phosphorothioate-modified rat p22phox antisense oligonucleotides to investigate the potential role of NAD(P)H oxidase. Expression of p22phox mRNA after Ang II-treatment was detected with Northern blots. Incorporation of [3H]leucine into de novo synthesized proteins was used as a parameter of cell hypertrophy. Expression of p27Kip1 was evaluated in cell lysates by Western blotting. RESULTS: Ang II stimulated the accumulation of O2. in tubular cells; however, an addition of two different antioxidants completely abolished measurable O2. This effect was transduced by angiotensin receptor type-1 (AT1) and was inhibited by a flavoprotein inhibitor (DIP) or p22phox antisense oligonucleotides, indicating the involvement of membrane NAD(P)H oxidase. Ang II-stimulated de novo protein synthesis was attenuated by DIP, antioxidants, and p22phox antisense oligonucleotides. The Ang II-induced expression of p27Kip1 protein and cellular hypertrophy were reduced by similar treatments. Generation of O2. by xanthine supplementation also stimulated p27Kip1 expression and induced hypertrophy in LLC-PK1 cells. CONCLUSIONS: This study provides the first evidence, to our knowledge, that Ang II induces O2. in cultured tubular cells. Ang II-mediated activation of membrane bound NAD(P)H oxidase, probably by an increase in p22phox transcripts, is likely responsible for this induction. Generation of O2. subsequently induces p27Kip1 expression and stimulates hypertrophy, suggesting a novel mechanism of how Ang II can modulate cell cycle regulation.

Angiotensin II↗
PubMed1998

Preimplantation diagnosis of autosomal dominant retinitis pigmentosum using two simultaneous single cell assays for a point mutation in the rhodopsin gene.

A couple requested preimplantation genetic analysis for a dominant form of retinitis pigmentosum caused by a C-->A transversion in the rhodopsin gene. Since this point mutation does not alter a restriction endonuclease site we designed two separate analytical systems, one involving site-specific mutagenesis and the other involving allele-dependent length polymorphism. After establishing the accuracy and robustness of these assay systems we utilized both systems simultaneously in a heminested polymerase chain reaction (PCR) system. This allowed accurate preimplantation diagnosis to be performed. One embryo was transferred but a pregnancy did not occur.

Cells, Cultured↗
PubMed1998

Glomerular expression of p27Kip1 in diabetic db/db mouse: role of hyperglycemia.

Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy.

Animals↗
PubMed1998

Release of stored retinol from adipocytes.

Adipose tissue is second only to liver as a retinol depot. Retinol is stored there is part as retinyl esters. In cultured mouse adipocytes, a hormone-sensitive lipase hydrolyzes these esters upon cAMP stimulation, with free retinol being released into the medium.

Adipocytes↗
PubMed1998

Vitamin A functions in the regulation of the dopaminergic system in the brain and pituitary gland.

Retinoic acids, bound to their receptors, function in the brain and pituitary gland by regulating the expression of the dopamine receptor D2R, which is a component of the dopaminergic system. The liganded retinoic acid receptors are specific transcription factors that are essential for full expression of D2R. D2R-knockout mice lack these retinoic acid receptors and exhibit a defect in locomotor ability similar to that seen in Parkinson's disease.

Animals↗
PubMed1998

Orexins: a newly discovered family of hypothalamic regulators of food intake.

A family of neuropeptides from the hypothalamus, named orexins, was recently discovered and characterized. Orexins stimulate appetite and food consumption. Their genes are expressed bilaterally and symmetrically in the lateral hypothalamus, which was earlier determined to be the "feeding center" of the hypothalamus. In contrast, the so-called satiety center is expressed in the ventromedial hypothalamus and is dominated by the leptin-regulated neuropeptide network.

Appetite↗