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M Urbanek

Publications and source records attributed to M Urbanek.

33 records · Page 2Linked to original sources
PubMed1996

Divergent effects of alpha 1-antitrypsin on the regulation of iron metabolism in human erythroleukaemic (K562) and myelomonocytic (THP-1) cells.

The acute-phase protein alpha 1-antitrypsin (alpha 1-AT) has been shown to inhibit the binding of transferrin to its cell-surface receptor. Here we demonstrate that in human erythroleukaemic cells (K562) alpha 1-AT enhances the binding affinity of iron-regulatory protein (IRP), the central regulator of cellular iron metabolism, to iron-responsive elements. Activation of IRP by alpha 1-AT is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and enhanced cell-surface expression of transferrin-binding sites, whereas ferritin production is decreased, although ferritin mRNA levels remain unchanged. In agreement with the well-established mechanism of cellular iron regulation, alpha 1-AT seems to modulate trf-rec and ferritin expression primarily post-transcriptionally/translationally by influencing IRP activity. In contrast, alpha 1-AT produces only minor changes in IRP activity, and subsequently in trf-rec expression and ferritin synthesis in THP-1 cells. Moreover the effects of alpha 1-AT on iron homeostasis in K562 cannot be overcome by the addition of iron salts, whereas concomitant treatment of THP-1 with iron and alpha 1-AT results in the same metabolic changes as the addition of iron alone. Because alpha 1-AT blocks transferrin binding on K562 as well as on THP-1 cells, it is suggested, on the basis of the results presented here, (1) that erythroid and monocytic cells might differ in their dependence on transferrin-mediated iron supply and (2) that THP-1 might be able to acquire iron by a transferrin-independent iron uptake system. alpha 1-AT might therefore be involved in the diversion of iron traffic between various cellular compartments under inflammatory conditions.

Cell Line↗
PubMed1996

The apportionment of dinucleotide repeat diversity in Native Americans and Europeans: a new approach to measuring gene identity reveals asymmetric patterns of divergence.

The purpose of this paper is to assess the extent of gene identity and differentiation at 33 dinucleotide repeat loci (377 total alleles) within and among three European and three Native American populations. In order to do this, we show that a maximum-likelihood method proposed for phylogenetic trees (Cavalli-Sforza and Piazza 1975) can be used to estimate gene identity (Nei 1987) with respect to any hierarchical structure. This method allows gene differentiation to be evaluated with respect to any internal node of a hierarchy. It also allows a generalization of F- and G-statistics to situations with unequal expected levels of differentiation. Our principal finding is that levels of genetic differentiation are unique to specific populations and levels of nesting. The populations of European origin show very little internal differentiation; moreover, their continental average is close to the total population defined by the aggregate of Europeans and Native Americans. By contrast, the Native American populations show moderate levels of internal differentiation, and a great distance between their continental average and the total. The results of analyses of subsets of loci that were selected to have high gene diversities in either Europeans or Native Americans closely parallel those from the total set of loci. This suggests that the principal results are unlikely to be caused by a European ascertainment bias in locus selection. In summary, our findings demonstrate that partitions of gene diversity into within- and between-populations components are heavily biased by the populations analyzed and the models fitted. Optimistically, however, more information is available to analyze population history and evolution by quantifying, as we have done, the uniqueness of patterns of differentiation.

Base Sequence↗
PubMed1996

Vulval Crohn's disease: difficulties in diagnosis.

Four patients with chronic vulval inflammation are described. The histological features of non-caseating granulomata and multinucleated giant cells are compatible with Crohn's disease, but only two patients had proven gastrointestinal involvement. The clinical and histological characteristics of Crohn's disease and other granulomatous inflammations of the vulva are discussed and the literature is reviewed.

Adult↗
PubMed1995

Polymorphism and genetic mapping of the human oxytocin receptor gene on chromosome 3.

Centrally administered oxytocin has been reported to facilitate affiliative and social behaviours, in functional harmony with its well-known peripheral effects on uterine contraction and milk ejection. The biological effects of oxytocin could be perturbed by mutations occurring in the sequence of the oxytocin receptor gene, and it would be of interest to establish the position of this gene on the human linkage map. Therefore we identified a polymorphism at the human oxytocin receptor gene. A portion of the 3' untranslated region containing a 30 bp CA repeat was amplified by polymerase chain reaction (PCR), revealing a polymorphism with two alleles occurring with frequencies of 0.77 and 0.23 in a sample of Caucasian CEPH parents (n = 70). The CA repeat polymorphism we detected was used to map the the human oxytocin receptor to chromosome 3p25-3p26, in a region which contains several important genes, including loci for Von Hippel-Lindau disease (VHL) and renal cell carcinoma.

Alleles↗
PubMed1995

Detection of hepatitis C viral sequences in serum by 'nested' polymerase chain reaction (PCR) and a commercial single-round PCR assay.

BACKGROUND: Demonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed. OBJECTIVE: The aim of the present study was to compare the new Amplicor assay with an 'in-house' PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified. STUDY DESIGN: Sera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h. RESULTS: A total of 52 patients were found to be positive for anti-HCV by the third-generation EIA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay. CONCLUSION: Because of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.

Journal Article↗
PubMed1995

An E-M algorithm and testing strategy for multiple-locus haplotypes.

This paper gives an expectation maximization (EM) algorithm to obtain allele frequencies, haplotype frequencies, and gametic disequilibrium coefficients for multiple-locus systems. It permits high polymorphism and null alleles at all loci. This approach effectively deals with the primary estimation problems associated with such systems; that is, there is not a one-to-one correspondence between phenotypic and genotypic categories, and sample sizes tend to be much smaller than the number of phenotypic categories. The EM method provides maximum-likelihood estimates and therefore allows hypothesis tests using likelihood ratio statistics that have chi 2 distributions with large sample sizes. We also suggest a data resampling approach to estimate test statistic sampling distributions. The resampling approach is more computer intensive, but it is applicable to all sample sizes. A strategy to test hypotheses about aggregate groups of gametic disequilibrium coefficients is recommended. This strategy minimizes the number of necessary hypothesis tests while at the same time describing the structure of disequilibrium. These methods are applied to three unlinked dinucleotide repeat loci in Navajo Indians and to three linked HLA loci in Gila River (Pima) Indians. The likelihood functions of both data sets are shown to be maximized by the EM estimates, and the testing strategy provides a useful description of the structure of gametic disequilibrium. Following these applications, a number of simulation experiments are performed to test how well the likelihood-ratio statistic distributions are approximated by chi 2 distributions. In most circumstances the chi 2 grossly underestimated the probability of type I errors. However, at times they also overestimated the type 1 error probability. Accordingly, we recommended hypothesis tests that use the resampling method.

Algorithms↗
PubMed1994

Alternative splice site selection in the human growth hormone gene transcript and synthesis of the 20 kDa isoform: role of higher order transcript structure.

Expression of the human growth hormone (hGH) gene in somatotrophs of the anterior pituitary gland results in the synthesis and secretion of a major 22 kDa and a minor 20 kDa GH isohormone. The expression of these two proteins reflects the alternative utilization of a major (B) and a minor (B') splice acceptor site in exon 3 of the hGH-N transcript. By comparing the structure and splicing patterns of the hGH-N gene transcript with that of the structurally related, placentally expressed, hGH-V gene transcript, which uses only the major (B) exon 3 splice acceptor, it has been possible to define the cis-acting elements in exon 3 that are critical for activation of the B' splice acceptor. The present paper demonstrates that, in addition to the importance of sequences in the immediate proximity of the two alternative splice acceptor sites, additional more remote sequences in the transcript also contribute to this alternative splice site selection. The data further suggest that these more distal sequences do not act individually, but interact so that the net level of alternative splicing in exon 3 is dictated by the overall higher order structure of the hGH-N transcript.

Alternative Splicing↗
PubMed1993

Functional characterization of the alternatively spliced, placental human growth hormone receptor.

The human growth hormone family of peptide hormones is encoded by five genes, pituitary growth hormone (hGH-N), and four placentally expressed genes, growth hormone variant (hGH-V), chorionic somatomammotropin A and B (hCS-A, hCS-B), and prolactin (hPrl). As part of an effort to define the local effects of the placentally expressed members of the GH/Prl family of hormones on the placenta, we have identified an isoform (hGHRd3) of the growth hormone receptor expressed in the placental villi. hGHRd3 mRNA differs from the liver GHR mRNA by the deletion of a 66-base pair segment encoding exon 3. In this study we show that hGHRd3 mRNA encodes a stable and functional receptor. hGHRd3 mRNA is efficiently translated and processed in a rabbit reticulocyte lysate translation system as well as in an in vivo Xenopus laevis oocyte expression system. In Xenopus oocytes hGHRd3 is stably integrated into the cell membrane and binds and internalizes ligand as efficiently as hGHR. hGHRd3 binds all three of the placentally expressed members of the GH/Prl gene family (hGH-V, hCS, and Prl) as well as both the 22 and 20 kDa isoforms of the pituitary hGH-N. The results of the present study strongly support the expression of a functional hGHRd3 isoreceptor in the placenta which may serve in autocrine, paracrine, and/or endocrine activation.

Animals↗
PubMed1992

Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript.

Four members of the GH gene family, human (h) GH-V, human chorionic somatomammotropin-A (hCS-A), hCS-B, and hCS-L, are expressed in the human placenta. In attempting to define the role of these hormones in placental development, we have structurally characterized the human placental GH receptor (GHR) mRNA. Human GHR cDNAs were cloned from a human placental cDNA library. Clone pGHR-P1 encompasses the entire hGHR-coding region and is identical to the previously reported liver hGHR cDNA, except for a precise deletion of the sequences corresponding to exon 3. This mRNA with an exon 3 deletion is the sole form of the hGHR mRNAs in the placental villi. A reverse transcription/polymerase chain reaction assay was used to further characterize GHR mRNA expression. Human GHR mRNA was detected in all placental tissues as well as in a wide spectrum of other tissues and cell lines. The distribution of the exon 3-retaining and exon 3-excluding isoforms of the hGHR mRNA shows distinct tissue specificity. Some tissues and cell lines contain only one of the two forms, and some contain a mixed population. Since exon 3 encodes a segment in the extracellular domain of the receptor, its alternative inclusion or exclusion may mediate critical alterations in hormone binding and physiological function.

Amino Acid Sequence↗
PubMed1990

Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif.

A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe. One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested. The sequence of the full-length cDNA was determined. An extended open reading frame predicted an encoded protein product of 20.5 kDa. This was directly confirmed by the in vitro translation of a synthetic mRNA transcript. Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers. The first and third fingers are of the C4 class while the second and fourth are of the C2HC class. Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit. Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man. The characteristics of this protein suggest that it serves a fundamental role in cellular function.

Amino Acid Sequence↗
PubMed1989

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Animals↗
PubMed1985

Asymptomatic decreased activities of hepatic glucose-6-phosphatase and glycogen phosphorylase in a number of children with chronic liver disease.

The evaluation of hepatic degradation of glycogen in patients with different chronic liver diseases was carried out on the basis of: a) specific activities of hepatic enzymes involved in catabolism of glycogen; b) level of glycogen in liver biopsies; c) concentration of glucose and cAMP in serum after the intravenous administration of glucagon. In 13 out of 35 patients investigated the activity of glucose-6-phosphatase was decreased to 14-50% of the control value. In the livers of 3 patients glycogen phosphorylase activity was decreased to 10% of the control value. In patients with the significantly low activities of hepatic glucose-6-phosphatase and phosphorylase a, however, normal catabolism of glycogen in the liver was observed, neither hypoglycemia nor abnormal glycogen storage in liver biopsies nor abnormal response to glucagon being found. In the group of patients with decreased and normal activities of glucose-6-phosphatase and phosphorylase a, biochemical parameters in the serum (i.e. markers of liver damage) were not detectable. Possible causes of the selective and asymptomatic decrease in the activities of glucose-6-phosphatase and phosphorylase a are discussed.

Adolescent↗
PubMedDate not supplied

A long-term toxicity test comprising reproduction and growth of zebrafish with 4-chloroaniline.

This study describes a long-term test over three generations, using zebrafish (Brachydanio rerio) as the test species and concentrations of 1, 0.2, and 0.04 mg/L 4-chloroaniline (CA) as a model substance. The effect of the compound on the ecologically important parameters reproduction and growth was the focus of interest. Reduction in egg release by fish raised under CA was the most sensitive parameter in the test. Compared to the toxic threshold concentration for growth (0.4 mg/L), egg release was affected by a ten-fold lower concentration (0.04 mg/L). This study demonstrates that a long-term test is still the most appropriate method to assess the chronic toxicity of a substance on fish. A chronic toxicity test is proposed which comprises two generations, with the zebrafish as test species.

Aniline Compounds↗