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Biomedical subjects

S Frank

Publications and source records attributed to S Frank.

At least 91 records · Page 5Linked to original sources
PubMed1998

ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6.

ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes.

ADP-Ribosylation Factor 1↗
PubMed1998

Multiple intracerebral haemangioblastomas in identical twins with von Hippel-Lindau disease--a clinical and molecular study.

Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant neoplastic disorder causing central nervous system haemangioblastomas. The VHL gene (3p25-3p26) is known to be a tumour suppressor gene, with its inactivation being responsible for a predisposition to tumour development. As far as we know, the present report of VHL disease manifestation in identical twins is unique. Genetic inquiry into the family background did not reveal this disease among their progenitors. For presymptomatic diagnosis of 17 presently unaffected family members, constitutional DNA of the twins was screened for VHL germline mutations, using loss of heterozygosity studies and exon-specific DNA sequencing. To determine the influence of somatic mutations of the VHL gene in tumourigenesis, DNA of five surgically removed intracerebral haemangioblastomas of the identical twins was analyzed in comparison with their constitutional DNA by DNA sequencing of the complete VHL coding region. However, no allelic losses were found for the VHL gene or for various other tumour suppressor genes (p53, BRCA1, BRCA2, DCC, and MCC). Furthermore, no mutations were found in the constitutional DNA of either twin sister or in the DNA of all five tumour lesions. Based on our observations, we conclude that in certain VHL families, presymptomatic molecular diagnosis of the disease is not feasible and requires close clinical surveillance of all individuals at risk.

Adult↗
PubMed1998

Decline in the expression of copper/zinc superoxide dismutase in the kidney of rats with endotoxic shock: effects of the superoxide anion radical scavenger, tempol, on organ injury.

1. Endotoxaemia causes an enhanced formation of reactive oxygen species (ROS) which contribute to the multiple organ dysfunction syndrome (MODS) in septic shock. Here we investigate (i) the effects of endotoxin on the expression of two isoforms of superoxide dismutase (SOD), namely Cu/Zn-SOD (cytosol) and Mn-SOD (mitochondria) in the rat kidney, and (ii) the effects of the radical scavenger tempol on the MODS caused by lipopolysaccharide (LPS, E. coli, 6 mg kg(-1) i.v.) in the rat. 2. Endotoxaemia resulted in a rapid, but transient, decline in the expression of both mRNA and protein of Cu/Zn-SOD as well as an increase in the expression of the mRNA of Mn-SOD in the kidney. Endotoxaemia for 6 h also caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of nitrite/nitrate. 3. Pretreatment of rats with tempol (100 mg kg(-1) i.v. bolus injection, 15 min prior to LPS followed by an infusion of 30 mg kg(-1) i.v., n=9) did not affect the circulatory failure, but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LPS. Tempol did not affect the rise in nitrite/nitrate caused by endotoxin. 4. These results imply that an enhanced formation of ROS (including superoxide anions) in conjunction with inadequate defences against such ROS contributes to the injury and dysfunction of the kidney and the liver in endotoxic shock.

Animals↗
PubMed1998

Coordinated induction of inducible nitric oxide synthase and GTP-cyclohydrolase I is dependent on inflammatory cytokines and interferon-gamma in HaCaT keratinocytes: implications for the model of cutaneous wound repair.

Recently we demonstrated a strong expression of inducible nitric oxide synthase (iNOS) and GTP-cyclohydrolase I (GTP-CH I) in the basal keratinocytes of the epidermis adjacent to the wound and of the hyperproliferative epithelium during wound healing. To identify possible mediators of iNOS and GTP-CH I expression during this process, we analyzed the regulation of iNOS and GTP-CH I expression in cultured human keratinocytes. We found a large and long lasting coinduction of iNOS and GTP-CH I expression upon simultaneous treatment of quiescent cells with inflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, but not with serum growth factors. The stimulatory effect of interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma is strongly synergistic on iNOS and GTP-CH I expression, because these factors alone stimulated GTP-CH I expression, although to a much lesser extent. Furthermore, iNOS mRNA levels are not influenced at all by stimulation with IL-1beta and revealed only a weak induction after treatment with tumor necrosis factor-alpha and interferon-gamma. Induction of iNOS and GTP-CH I gene expression upon cytokine and interferon-gamma exposure is independent of de novo protein synthesis. Because these cytokines are present at the wound site, they might be responsible for iNOS and GTP-CH I induction during cutaneous repair. Serum, which is released upon hemorrhage, is likely to play no stimulatory role in iNOS and GTP-CH I induction during wound healing.

Antioxidants↗
PubMed1998

Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.

Recent work has suggested a possible role of nitric oxide, a free radical gas, during the wound healing process. In this study we investigated the regulation of inducible nitric oxide synthase (iNOS) and GTP-cyclohydrolase I (GTP-CH I), the rate-limiting enzyme in the biosynthesis of the iNOS cofactor (6R) 5,6,7,8-tetrahydrobiopterin (6-BH4), during the repair process. We found a similar time course of induction of iNOS and GTP-CH I expression, whereas absolute expression levels were different for both genes. Immunohistochemical analysis revealed colocalization of iNOS and GTP-CH I proteins in the wound. Systemic treatment with glucocorticoids significantly altered the expression levels of iNOS and GTP-CH I. Expression of iNOS and GTP-CH I was suppressed by glucocorticoids in normal, and to a much greater extent in wounded skin. Furthermore, a role of nitric oxide as a novel mediator of gene regulation during healing is suggested by the demonstration of nitric oxide-mediated induction of vascular endothelial growth factor expression in keratinocytes. These findings may provide an explanation for the beneficial effects of orally supplemented L-arginine on wound healing, and suggest that a disturbed induction of iNOS and GTP-CH I expression may at least partially underlie the wound healing defect seen in glucocorticoid-treated animals.

Animals↗
PubMed1998

Mouse HNF-3/fork head homolog-1-like gene: structure, chromosomal location, and expression in adult and embryonic kidney.

Screening of a mouse kidney cDNA library with a HNF-3/fork head domain probe revealed cDNA Hfh-1L containing the highly conserved fork head DNA-binding domain. The Hfh1L cDNA shows 92.7% homology at the nucleic acid level with the fork head gene HFH-1 from rat. Southern blot analyses demonstrated that the Hfh-1L gene is highly conserved in a wide variety of species, including goldfish and frog. Sequencing the corresponding genomic clone, we found that the Hfh-1L gene is most likely intronless. By interspecific back-cross analysis, the Hfh-1L gene was localized to mouse chromosome 13. In order to analyze the expression pattern of Hfh-1L, we performed Northern blot analyses and revealed a 2.7-kb transcript in adult kidney and stomach. In situ hybridization experiments of adult mouse kidney showed Hfh-1L expression in the outer medulla of the kidney and the transitional epithelium. In light of the significance of a number of fork head genes in early embryonic development, the pattern of expression during murine embryogenesis was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), and Hfh-1L transcripts were detected in mouse embryos at every stage tested from day 10.5 to 16.5 postconception (p.c.) and in the developing metanephros of 14.5- and 15.5-day p.c. embryos. This expression pattern suggests that the Hfh-1L gene is involved in the development of the kidney.

Amino Acid Sequence↗
PubMed1998

Identification of a differential expression of two cDNAs between malignant mesothelioma and normal mesothelial cells using the RNA fingerprint method.

The RNA fingerprint method was used to identify mRNAs that were differentially expressed during the development of human mesothelial cell cancer. We report the isolation of two differentially expressed clones. One clone was expressed in the metastatic mesothelioma cell line M1A and in the malignant mesothelioma cell line M1 and downregulated in normal mesothelial cells. M1 and M1A were derived from a primary and metastatic tumor of the same patient. The other clone was only expressed in normal mesothelial cells. The different expression pattern was confirmed by Northern blot analysis. One clone had a striking sequence homology to the soares pregnant uterus NbHPU homo sapiens cDNA clone. The other clone contained a high sequence homology to the human mRNA for ORF. The biological function of the corresponding genes is unknown. The specificity of expression of the two sequence tags was further examined on different cancer cell lines and normal tissues.

Base Sequence↗
PubMed1998

Failure of reducing lower extremity amputations in diabetic patients: results of two subsequent population based surveys 1990 and 1995 in Germany.

BACKGROUND: A 50% reduction of lower extremity amputations during the subsequent 5 year period has been targeted by the St. Vincent-Declaration issued in 1989/90 for a better care of diabetic patients across Europe. PATIENTS AND METHODS: In two adjacent counties far off major city areas 10 hospitals without specialised diabetes centers in the area provide care to about 300,000 inhabitants. Based on the official operation books and verified by the individual patient file all patients amputated in the 10 hospitals during the years 1990 and 1995 were evaluated retrospectively. RESULTS: A total of 119 patients (66 males, 53 females, age median 72 years) were amputated in the 10 hospitals 1990, and 162 (89 males, 73 females, age median 74 years) in 1995. The proportion of diabetic amputees amounted to 70.6 and 62.3%, respectively. A trend towards more toe amputations in diabetic versus nondiabetic patients was seen in both surveys which reached significance in 1995 (59 vs. 41%; p < 0.05). Based on the total population and the estimated number of diabetic patients (5% of the population) 1.4 and 2/10,000 nondiabetics were amputated in 1990 and 1995, respectively, in contrast to 61 and 66/10,000 diabetic individuals, indicating a 44 fold and 33 fold excess risk of diabetic patients. CONCLUSION: It is concluded that these 2 surveys 5 years apart reveal a failure of reducing lower extremity amputations in people with diabetes--despite the objectives of the St. Vincent-Declaration.

Adolescent↗
PubMed1997

A novel type of glutathione peroxidase: expression and regulation during wound repair.

We have previously identified and cloned a novel keratinocyte growth factor (KGF)-regulated gene in human keratinocytes that encodes the human homologue of a bovine non-selenium glutathione peroxidase (GPx). To gain insight into the regulation of this gene in vivo, we isolated the murine homologue from a mouse skin cDNA library. In vitro transcription/translation demonstrated that the cDNA encodes a 27 kDa protein. Furthermore, we amplified by PCR a partial cDNA that most likely corresponds to a related gene. RNase protection analysis revealed tissue-specific expression of both genes and the occurrence of alternative splicing or RNA editing of at least one of the primary transcripts. Similar to that of KGF, expression of GPx was strongly induced after cutaneous injury, and each isoform displayed unique kinetics of expression during the repair process. In situ hybridization studies demonstrated high levels of GPx mRNA in keratinocytes of the hyperproliferative epithelium at the wound edge. Since these cells express functional KGF receptors, induction of GPx expression by KGF might also occur in vivo. These data suggest a role for GPx in the protection of epithelial cells against oxidative stress, particularly during the inflammatory phase of wound repair.

Amino Acid Sequence↗
PubMed1997

The putative tumor suppressor gene FHIT at 3p14.2 is rarely affected by loss of heterozygosity in primary human brain tumors.

To elucidate the role of the recently identified FHIT gene, located at 3p14.2 in human brain tumor carcinogenesis, a total of 259 tumors were analyzed for loss of heterozygosity (LOH) at microsatellite loci D3S1313, D3S1234, D3S1300, and D3S1481. In primary brain tumors, LOH was detected at a frequency of 8.4% (n = 214). Low-grade gliomas exhibited insignificantly lower LOH rates in comparison to high-grade gliomas (5.3%, n = 19, versus 11.1%, n = 90). Notably, no allelic loss was observed in 12 recurrent glioblastomas analyzed in comparison to their corresponding primary tumor lesions and in two astrocytomas with progression to higher grades of malignancy. Our data indicate that allelic loss of the FHIT gene is neither a critical event in carcinogenesis of primary brain tumors nor tumor grade-associated in astrocytic tumors. In contrast, observed LOH rate for brain metastases was as high as 54.5% (n = 45), in accordance with data thus far accumulated from analyses of corresponding primary tumors.

Acid Anhydride Hydrolases↗
PubMed1997

The human homologue of a bovine non-selenium glutathione peroxidase is a novel keratinocyte growth factor-regulated gene.

Keratinocyte growth factor is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin. Furthermore, it has been implicated in morphogenetic processes of several organs. To further define the mechanisms of KGF action in the skin, we attempted to identify genes which are regulated by KGF in keratinocytes. Using the differential display RT-PCR technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines. Molecular cloning of the full-length cDNA revealed a strong homology of the corresponding gene product with a bovine non-selenium glutathione peroxidase. Upon transfection of COS cells with the full-length cDNA, a 27 kD cytoplasmic protein was obtained which had the expected size of glutathione peroxidase. Expression of the novel gene was detected in normal human skin and at particularly high levels in psoriatic skin, indicating a possible in vivo function of the protein in this tissue. Identification of a peroxidase as a KGF-regulated gene suggests that prevention from oxygen toxicity is a novel and specific mechanism of KGF action.

Amino Acid Sequence↗
PubMed1997

An intronic germline transition in the HNPCC gene hMSH2 is associated with sporadic colorectal cancer.

The aim of this study was to determine whether an intronic germline substitution in the hereditary non-polyposis colorectal cancer (HNPCC) gene hMSH2 represents a genetic risk factor for sporadic CRC. Possible effects of this substitution were investigated by assessment of microsatellite instability and hMSH2 cDNA sequencing. Constitutional DNA from patients with sporadic CRC and healthy controls from the same region in Germany was analysed for the intronic germline T-->C transition six bases upstream of exon 13 of hMSH2. 29 of 106 patients (27%) were found to harbour the germline T-->C transition as opposed to only 13 of 125 controls (10%; P < 0.001; OR 3.2, CI 1.58-6.63). CRCs from patients with the substitution displayed neither clinical HNPCC-like features nor an increased rate of microsatellite instability. No abnormal cDNA sequence was found at the exon 12-13 border. These data suggest a 3.2-fold increased risk of sporadic CRC for individuals with the intronic hMSH2 transition. However, this substitution might not be pathogenic itself, but may be linked to a locus nearby that is.

Adult↗
PubMed1997

The role of apo-(a) kringle-IVs in the assembly of lipoprotein-(a).

Lipoprotein-(a) [Lp(a)] is a highly atherogenic lipoprotein with unknown function, consisting of a low-density lipoprotein (LDL) core and the apo(a) glycoprotein. The characteristic structural feature of apo(a) is the presence of multiple so called "kringle' repeats which are in part identical and in part exhibit slight sequence differences. The assembly of apo(a) and LDL, which is determinant for plasma Lp(a) levels, takes place extracellularly and requires specific structural motifs in apo(a) and apoB. Here we studied the structural features in apo(a) necessary for high-efficient assembly. Thirteen recombinant apo(a) glycoproteins, which differed in the set of kringle-IV (K-IV) motifs, were expressed in COS-7 cells and incubated with LDL. The rate of total and disulfide-stabilized Lp(a) complex formation was measured by an immunochemical assay. Constructs containing K-IV T(type)5-T10 yielded almost 100% total and 80% stable complexes, respectively. Deletion or replacement of the different kringles revealed that K-IV T6 and T7 were responsible for the high-yield assembly and that K-IV T5 had an amplifying effect. Increasing the absolute number of K-IV repeats had an additional amplifying effect. The rate of Lp(a) assembly correlated strongly with the affinity of these constructs to Lys-Sepharose. Our results have implications for understanding the metabolism of Lp(a) and may help to design strategies for searching natural apo(a) mutants with aberrant plasma Lp(a) levels.

Animals↗
PubMed1997

Metabolism of Lp(a): assembly and excretion.

Lp(a) is one of the most atherogenic lipoproteins, and we know much more about the pathophysiology of Lp(a) than about its physiological function and metabolism. From our previous investigations and the new results reported here, we propose the following model of Lp(a) metabolism: apo(a) is biosynthesized in liver cells and the size of the isoform determines its rate of synthesis and excretion. Specific kringle-4 domains in apo(a), mainly T-6 and T-7, bind in a first step to circulating LDL, followed by the stabilization of the newly formed Lp(a) complex by a disulfide bridge. Circulating Lp(a) interacts specifically with kidney cells, or possibly other tissues, causing cleavage of 2/3-3/4 of the N-terminal part of apo(a) by a collagenase-type protease. Part of the apo(a) fragments is found in the urine, but there are indications that they in fact represent the biologically active form of apo(a). The core portion of Lp(a) in turn is cleared by the LDL-receptor or another specific binding system of the liver. Strategies for reducing plasma Lp(a) levels with medication should aim at interfering with the assembly of Lp(a) on one hand and the stimulation of apo(a) fragmentation on the other hand.

Animals↗
PubMed1996

The human homologue of the yeast CHL1 gene is a novel keratinocyte growth factor-regulated gene.

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin. To gain insight into the mechanisms of KGF action in this tissue, we attempted to identify genes that are regulated by KGF in keratinocytes. Using the differential display reverse transcription polymerase chain reaction technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines. This gene seems to be part of a multigene family as assessed by Southern blot analysis. Molecular cloning and sequencing of the full-length cDNA revealed a strong homology with the yeast CHL1 gene. The latter encodes a putative helicase, which is involved in correct chromosome transmission and cell cycle progression. Furthermore, the CHL1 gene product and the protein encoded by the novel KGF-regulated gene were identical in size, indicating that we had cloned the human CHL1 homologue. This finding suggests a novel and specific role of KGF in correct chromosome segregation and/or cell cycle progression.

Amino Acid Sequence↗