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Biomedical subjects

A Umar

Publications and source records attributed to A Umar.

36 records · Page 2Linked to original sources
PubMed1997

A preliminary comparison of plasma fibrinogen concentrations, leukocyte numbers and erythrocyte sedimentation rate as non-specific indicators of inflammatory conditions in buffalo (Bubalis bubalis).

The plasma fibrinogen concentration (Fib), total leukocyte count (TLC), neutrophil, lymphocyte and monocyte numbers, and the erythrocyte sedimentation rate (ESR) were determined in 153 buffaloes suffering from different clinical conditions. Fib increased significantly (p < 0.05) in chronic mastitis, pyrexia, pyometra, cutaneous abscesses, tail gangrene and acute indigestion, whereas in most of the other conditions studied it varied non-significantly. TLC increased significantly in chronic mastitis, pyrexia, endometritis, cutaneous abscesses and infected skin wounds. An increase in neutrophils was associated with an increased TLC. Numbers of lymphocytes varied non-significantly in most of the conditions. Monocytes decreased significantly in most of the acute conditions. ESR was significantly elevated in all clinical conditions. Significantly increased mean Fib values in the different conditions varied from 703 +/- 119 to 725 +/- 140 mg/dl, while the maximum individual value was 1510 mg/dl in a case of cutaneous myiasis. The significantly increased mean TLC ranged from 9.48 +/- 2.91 to 11.1 +/- 3.5 x 10(3)/microliter, while it was 21.7 x 10(3)/microliter in a case of meningitis. ESR values in sick buffaloes varied from 57 to 111 mm in the first hour.

Animals↗
PubMed1996

Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis.

A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Meiotic pachytene arrest in MLH1-deficient mice.

Germ line mutations in DNA mismatch repair genes including MLH1 cause hereditary nonpolyposis colon cancer. To understand the role of MLH1 in normal growth and development, we generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals that the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis.

Adaptor Proteins, Signal Transducing↗
PubMed1996

DNA-replication fidelity, mismatch repair and genome instability in cancer cells.

It has been suggested that an early event in the multistep progression of a normal cell to a tumor cell could be a defect that leads to an elevated mutation rate, thus providing a pool of mutants upon which selection could act to yield a tumor. Such a mutator phenotype could result from a defect in any of several DNA transactions, including those that determine the DNA replication error rate or the ability to correct replication errors. Recent evidence for the latter is the mutator phenotype observed in tumor cells of patients having a hereditary form of colon cancer. These patients have a germline mutation in genes required for post-replication DNA mismatch repair. A second mutation arises somatically, yielding a greatly elevated mutation rate due to an inability to correct DNA replication errors. This connection between cancer, DNA replication errors and defective mismatch repair is the subject of this review, wherein we consider the key steps and principles for high fidelity replication and how their perturbation results in genome instability.

Base Sequence↗
PubMed1996

Mutation of MSH3 in endometrial cancer and evidence for its functional role in heteroduplex repair.

Many human tumours have length alterations in repetitive sequence elements. Although this microsatellite instability has been attributed to mutations in four DNA mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds, many sporadic tumours exhibit instability but no detectable mutations in these genes. It is therefore of interest to identify other genes that contribute to this instability. In yeast, mutations in several genes, including RTH and MSH3, cause microsatellite instability. Thus, we screened 16 endometrial carcinomas with microsatellite instability for alterations in FEN1 (the human homolog of RTH) and in MSH3 (refs 12-14). Although we found no FEN1 mutations, a frameshift mutation in MSH3 was observed in an endometrial carcinoma and in an endometrial carcinoma cell line. Extracts of the cell line were deficient in repair of DNA substrates containing mismatches or extra nucleotides. Introducing chromosome 5, encoding the MSH3 gene, into the mutant cell line increased the stability of some but not all microsatellites. Extracts of these cells repaired certain substrates containing extra nucleotides, but were deficient in repair of those containing mismatches or other extra nucleotides. A subsequent search revealed a second gene mutation in HHUA cells, a missense mutation in the MSH6 gene. Together the data suggest that the MSH3 gene encodes a product that functions in repair of some but not all pre-mutational intermediates, its mutation in tumours can result in genomic instability and, as in yeast, MSH3 and MSH6 are partially redundant for mismatch repair.

Base Sequence↗
PubMed1995

Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines.

The instability of short repetitive sequences in tumor DNA can result from defective repair of replication errors due to mutations in any of several genes required for mismatch repair. Understanding this repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the mismatch repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific mismatch repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The repair defects were independent of whether the signal for strand specificity, a nick, was 5' or 3' to the mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective mismatch repair and the suggestion that diminuition in mismatch repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of repair-proficient and repair-deficient cell lines for future studies of mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Microsatellite instability in gynecological sarcomas and in hMSH2 mutant uterine sarcoma cell lines defective in mismatch repair activity.

We have examined a panel of gynecological sarcomas for microsatellite instability. The genomic DNA from 11 of 44 sarcomas contained somatic alterations in the lengths of one or more di-, tri-, tetra-, or pentanucleotide microsatellite sequence markers, and 6 of these cases had alterations in two or more markers. In addition, di-, tri-, and tetranucleotide microsatellites were found to be highly unstable in single cell clones of two cell lines derived from a uterine mixed mesodermal tumor. Since such instability is characteristic of cells defective in postreplication mismatch repair, we examined mismatch repair activity in extracts made from these lines. Both extracts were repair deficient, while an extract of another gynecological sarcoma cell line not exhibiting microsatellite instability was repair proficient. The repair deficiency was complemented by a colon tumor cell extract that was defective in the hMLH1 protein but not by an extract defective in hMSH2 protein. This suggested that the defect in the uterine sarcoma line could be in hMSH2. Subsequent analysis of the gene revealed a 2-bp deletion in exon 14, leading to premature truncation of the hMSH2 protein at codon 796 and no detectable wild-type gene present. These data suggest that the microsatellite instability observed in these cell lines, and possibly in a significant number of gynecological sarcomas, is due to defective postreplication mismatch repair. There was no apparent correlation with microsatellite instability and clinical outcome.

DNA Repair↗
PubMed1995

Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint.

The human colon tumor cell line HCT116 is deficient in wild-type hMLH1, is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Transferring a normal copy of hMLH1 on chromosome 3 into the cell line restores MMR activity, stabilizes microsatellite loci, and increases the sensitivity of the cell to MNNG. Previous studies in other cell lines tolerant to alkylating agents such as MNNG or N-methylnitrosourea have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypothesis that tolerance to MNNG or 6TG may be the result of MMR deficiency. To test this hypothesis, we studied the effects of 6TG on the MNNG-tolerant, MMR-deficient HCT116 cell line and its MNNG-sensitive, MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Continuous exposure to low doses of 6TG (0.31-1.25 micrograms/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dose-dependent decrease in CFA. Growth kinetics and cell cycle analysis revealed that the growth of 6TG-treated HCT116 + chr3 cells was arrested at G2 after exposure to low dose of 6TG. In contrast, the same exposure to 6TG did not induce G2 arrest but rather a G1 delay in HCT116 and HCT116 + chr2. To obtain further evidence for the role of MMR on 6TG and MNNG toxicity, we isolated an MNNG-resistant revertant clone, M2, from the MNNG-sensitive, MMR-proficient HCT116 + chr3 cell line and characterized the MMR activity, hMLH1 status, and 6TG response. The results showed that M2 cells lost MMR activity as well as the previously introduced normal hMLH1 gene. Restoration of the CFA of M2 and an absence of G2 arrest were observed after treatment with low doses of 6TG. These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The results further suggest that any agent that induces DNA mispairs will cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells.

Azaguanine↗
PubMed1995

A hPMS2 mutant cell line is defective in strand-specific mismatch repair.

Human cells contain several homologs of the bacterial mutL gene required for mismatch repair, including a gene on chromosome 7 designated hPMS2. We have identified an endometrial carcinoma cell line, HEC-1-A, that has a C-->T mutation in hPMS2 that generates a nonsense codon and yields a protein truncated at the C terminus. No wild-type gene or gene product was detected. The missing amino acids in hPMS2 are highly conserved among PMS homologs, suggesting that they may be critical for function. In support of this, extracts of HEC-1-A cells are defective in repairing a variety of mismatched substrates. Moreover, di-, tri-, and tetranucleotide repeated sequences are highly unstable in single cell clones of HEC-1-A cells, and HEC-1-A cells are resistant to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The results provide strong experimental support for the involvement of the hPMS2 gene product in mismatch repair in human cells and support the concept that a defective hPMS2 gene may lead to predisposition to certain forms of cancer.

Adenosine Triphosphatases↗
PubMed1995

Reciprocal homologous recombination in or near antibody VDJ genes.

To determine if rearranged heavy chain variable (VDJ) genes can recombine with each other by crossing over of DNA strands, we constructed a transgene that contained a promoter, VDJ gene, reporter gene to detect crossover events, intron enhancer, matrix attachment region, and constant gene for IgM (C mu). Following immunization of transgenic mice, hybrid molecules were isolated from B cell DNA which contained the transgene recombined with the endogenous IgH locus. Reciprocal products of crossovers were detected by plasmid rescue and PCR amplification, and they were sequenced. Recombination occurred somewhere within 147 bp of homology that contained the JH4 gene segment and 3' flanking DNA. The recombined transgenes had a 20-fold increase in mutation in the VDJ region compared to nonrecombined transgenes, which indicates that DNA sequences 3' of the C mu gene in the endogenous IgH locus are necessary for full activity of the mutator mechanism. The recovery of recombinants between VDJ transgenes and endogenous VDJ genes raises the possibility that reciprocal recombination may somatically diversify rearranged genes between maternal and paternal alleles.

Animals↗
PubMed1994

DNA loop repair by human cell extracts.

An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5' or 3' to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type hMLH1 gene and is deficient in repair of mismatches. However, a cell line with deletions in both hMSH2 alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation.

The human colon tumor cell line HCT 116 is known to have a homozygous mutation in the mismatch repair gene hMLH1 on human chromosome 3, to exhibit microsatellite instability, and to be defective in mismatch repair. In order to determine whether the introduction of a normal copy of hMLH1 gene restores mismatch repair activity and corrects microsatellite instability, a single human chromosome 3 from normal fibroblasts was transferred to HCT 116 cells via microcell fusion. As a control, human chromosome 2 was also transferred to HCT 116 cells. Two HCT 116 microcell hybrid clones that received a single copy of chromosome 2 (HCT 116 + ch2) and two that received a single copy of chromosome 3 (HCT 116 + ch3) were isolated and characterized. A G-G mismatch in M13-derived heteroduplex DNA was efficiently repaired in cell extracts from HCT 116 + ch3 cells, but not in those of parent HCT 116 cells or HCT 116 + ch2 cells. Microsatellite alterations at the D5S107 locus containing CA repeats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 150 subclones from HCT 116 + ch2 cells. In contrast, none of the 225 subclones derived from mismatch repair-proficient HCT 116 + ch3 cells showed alterations in the microsatellite at the same locus. The effect of introducing chromosome 3 on the sensitivity of HCT 116 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined, since enhanced tolerance to MNNG is accompanied by loss of mismatch repair activity in several cell lines. Within 3 days after treatment with 5 microM MNNG, HCT 116 + ch3 cells became morphologically flat and stopped growing. Their colony-forming ability, determined 10 days after treatment, was reduced 200-fold when compared to MNNG-treated parental HCT 116 and HCT 116 + ch2 cells. These results support the hypothesis that mutations in both alleles of the hMLH1 gene are necessary for the manifestation of defective mismatch repair and microsatellite instability and for enhanced MNNG tolerance. The results also suggest that the mismatch repair system contributes to the process that causes growth arrest in response to DNA damage by alkylating agents.

Base Sequence↗
PubMed1994

Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability.

A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.

Cell Line↗
PubMed1994

Preliminary observations on plasma fibrinogen and plasma protein concentrations and on plasma protein:fibrinogen ratios in clinically healthy buffaloes.

The plasma fibrinogen concentration, the total plasma protein concentration and the plasma protein to fibrinogen ratio (PP:F) were determined in clinically healthy Nili Ravi buffaloes. The plasma fibrinogen concentrations in calves, lactating and non-lactating buffaloes were 513 +/- 62, 615 +/- 90 and 544 +/- 74 mg/dl, respectively, and were statistically different (p < 0.05). Total plasma protein concentrations in these animals were 7.15 +/- 0.28, 9.32 +/- 0.53 and 8.79 +/- 0.58 g/dl. PP:F for all animals was between 11 and 19. Fibrinogen levels were positively correlated with plasma protein (r = 0.59) and negatively correlated with PP:F (r = -0.59).

Animals↗
PubMed1992

Linkage of two pseudogenes from V kappa 1 and V kappa 9 murine immunoglobulin families.

As an initial step towards the molecular analysis of the murine V kappa locus, a cosmid library from BALB/cJ mouse liver DNA was screened with probes representing 10 V kappa families. Of eight cosmids that were isolated from the initial screen, five contained a single restriction fragment that hybridized to the probes. Two cosmids contained two fragments that hybridized to the same probe, V kappa 4, indicating that some V kappa 4 gene segments are linked. One cosmid had two genes that belonged to different families, V kappa 1 and V kappa 9. The two gene segments were located within 12 kb of each other and lay in the same transcriptional orientation. Linkage of gene segments from the V kappa 1 and V kappa 9 families is consistent with a genetic map of the locus, and provides physical evidence for the first time that two genes from different families are closely linked in the murine kappa locus. Sequence analysis revealed that both genes are pseudogenes: the V kappa psi 1.7 gene segment has eight mutations, including termination codons, insertions, and deletions, and the V kappa psi 9B.8 gene segment has two mutations of an insertion and altered RNA splice site. Both genes have the potential to rearrange based on the sequence of their heptamer-nonamer motifs. The identification of pseudogenes raises the question of how many nonfunctional genes are present in the murine germline repertoire.

Amino Acid Sequence↗
PubMed1991

Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.

Somatic mutation in immunoglobulin genes is localized to a 2-kilobase region of DNA surrounding and including rearranged variable (V), diversity, and joining (J) gene segments encoding heavy and light chains. To examine the structural basis for targeted mutation, we developed an assay to score mutation on plasmid substrates by using a reporter gene: a bacterial gene encoding an amber-suppressor tRNA molecule was placed 3' of a rearranged kappa VJ gene within the boundaries of mutation. The reporter gene is exquisitely suited for mutational analysis because it is only 200 base pairs (bp), which should not greatly disrupt structure of the immunoglobulin locus, and gene function depends on secondary structure, which means mutation can be scored in many different nucleotide positions. The plasmid was used to make transgenic mice, which were then immunized. The shuttle vector was retrieved by plasmid rescue into an indicator strain of Escherichia coli that contained an amber mutation in its beta-galactosidase gene. Integrity of the tRNA molecule was monitored by colony color, which permitted many transformants to be screened visually. Mutations were not seen in DNA from a transfected B-cell line grown in vitro or in DNA from nonlymphoid tissue of transgenic mice, indicating that the reporter gene was stable during cell division and DNA manipulations. However, when the transgenic mice were immunized, DNA from splenic B cells contained point mutations in the reporter gene at a frequency of 10(-3) per transformant. Sequence analysis of 17 mutated transgenes revealed that the mutations were 1- and 2-bp deletions in the tRNA gene, and one plasmid had an additional 2-bp deletion in the V gene. In contrast, previous studies have shown that mutations in endogenous VJ genes are predominantly nucleotide substitutions and have only 6% deletions. Two other plasmid constructs were analyzed in transgenic lines: no mutations were found when the tRNA gene was placed distal to the VJ gene, and no mutations were seen when the immunoglobulin promoter was deleted. Although we lack direct evidence that the deletions in the tRNA gene are caused by the same mechanism that acts on VJ genes, we have shown that mutations in this assay occur in a manner consistent with immunoglobulin-specific mutation in that they are found in splenic B cells and not in tail tissue, depend on position next to the VJ gene, and require transcription of the VJ gene.

Animals↗