Biomedical subjects

B Smit

Publications and source records attributed to B Smit.

At least 19 recordsLinked to original sources

PubMed2000

Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases.

Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These findings extend the conservation of the NER apparatus and the link between NER and basal transcription and suggest that hMMS19 exerts its function in repair and transcription by interacting with the XPB and XPD helicases.

Amino Acid Sequence↗

PubMed2000

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice.

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

Alleles↗

PubMed1999

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Acute Disease↗

PubMed1999

Associations between stomach cancer incidence and drinking water contamination with atrazine and nitrate in Ontario (Canada) agroecosystems, 1987-1991.

BACKGROUND: Nitrate and atrazine are two chemicals that are heavily used in certain sectors of agriculture. They are suspected to be associated with the development of certain types of tumours. METHODS: Existing data were obtained on the incidence of specific types of cancers, contamination of drinking water with atrazine and nitrate, and related agricultural practices for the 40 ecodistricts in the province of Ontario. The data were merged into a georelational database for geographical and statistical analyses. Weighted (by population size) least squares regression analyses were conducted while controlling for confounding socioeconomic and lifestyle factors. Maximum likelihood spatial error models were estimated when least square regression error terms were found to be spatially autocorrelated using the Moran's I statistic. RESULTS: Atrazine contamination levels (range 50-649 ng/l, maximum acceptable concentration [MAC] = 60000 ng/l) were positively associated (P < 0.05) with stomach cancer incidence and negatively associated with colon cancer incidence. Nitrate levels, (range 0-91 mg/l, MAC = 10 mg/l) were negatively associated with stomach cancer incidence. CONCLUSION: The associations found at the ecodistrict level, both positive and negative, if confirmed by other studies, raise serious questions about maximum allowable limits for atrazine, as well as possibilities of complex trade-offs among disease outcomes, and interactions of biophysical and social mechanisms which might explain them. Although the negative associations appear to have no direct biological explanations, such counter-intuitive outcomes may occur in complex systems where social and biological variables interact.

Adult↗

PubMed1998

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).

Amino Acid Sequence↗

PubMed1998

Assessment of the micronucleus assay as a biological dosimeter using cytokinesis-blocked lymphocytes from cancer patients receiving fractionated partial body-radiotherapy.

PURPOSE: To assess the suitability of the cytokinesis block micronucleus assay as a biological dosimeter following in-vivo radiation using cancer patients undergoing radiotherapy. METHODS: Blood from 4 healthy donors was irradiated in vitro with gamma-rays and the dose response of induced micronuclei in binucleate lymphocytes following cytokinesis block was determined. Micronucleus frequency was ascertained before and at intervals during radiotherapy treatment in 6 patients with various tumors in the pelvic region. Equivalent whole body doses (physical doses) at these times were calculated from radiation treatment plans and cumulative dose volume histograms. RESULTS: Linear dose response relationships were found for induced micronucleus frequency in lymphocytes resulting from both in-vitro and in-vivo irradiation. Doses resulting from in-vivo irradiation (biological doses) were estimated by substitution of micronucleus frequency observed in radiotherapy patients into the dose response curve from in-vitro irradiation of blood. The relationship between the biologically estimated dose (BD) and the calculated equivalent whole body dose (PD) was BD = 0.868 (+/- 0.043)PD + 0.117 (+/- 0.075). CONCLUSION: The micronucleus assay appears to offer a reliable and consistent method for equivalent whole body radiation dose estimation, although our findings should be confirmed using lymphocytes from radiotherapy patients with tumors at anatomical sites other than the pelvis. Except at doses lower than about ).4 Gy, the method yields dose estimates acceptably close to "true" physically determined doses. The assay can be performed relatively rapidly and can be used as a "first line" biological dosimeter in situations where accidental exposure to relatively high radiation doses has occurred.

Adult↗

PubMed1998

Exercise therapy for intermittent claudication: a review of the quality of randomised clinical trials and evaluation of predictive factors.

OBJECTIVE: To establish the effect of exercise therapy in patients with intermittent claudication and to identify outcome predictors for exercise training. DESIGN: A methodological study of randomised clinical trials. METHODS: A quality assessment of all eligible studies was performed, using a list of methodological criteria. A weighing scale for the criteria was developed, based on four main categories: study population, intervention, outcome variables and data presentation/analysis. RESULTS: Ten studies were included in the analysis. Seven randomised clinical trials had a methodological score of 60 or more points (maximum 100), and were considered to be of good quality. The mean of the methodological score was 62.5 (S.D. 8.5). Improvement in pain-free/maximum walking distance/time ranged from 28-210% (mean 105, S.D. 55.8). Only one study evaluated outcome predictors for exercise therapy. CONCLUSIONS: All studies reported a positive effect of exercise therapy on walking distance in patients with intermittent claudication, but no predictive factors were clearly identified. Future research efforts should focus on improving the quality of clinical research for patients with intermittent claudication and developing optimal rehabilitation programs.

Exercise Therapy↗

PubMed1998

The effect of exercises on walking distance of patients with intermittent claudication: a study of randomized clinical trials.

BACKGROUND AND PURPOSE: There is no consensus about the indication for exercises for patients with intermittent claudication of the lower extremity and the characteristics of an exercise program to improve walking distance. The effect of walking is assessed by a systematic review of randomized clinical trials. METHODS: Literature databases were accessed using the relevant key words. The references of identified articles were screened for additional studies. A checklist was developed to screen the studies with respect to the variables of interest. A methodological assessment form was developed to assess the methodological quality of the studies (maximum possible score: 100). RESULTS: Eighty-two articles were identified, of which 21 studies were considered relevant for inclusion in the review. Following the analysis of the articles, 11 studies were for various reasons eliminated, leaving 10 studies for the systematic review. The score for methodological quality of the studies ranged from 47 to 75 (mean = 62.5, SD = 8.5). Percentage of improvement in walking distance or time ranged from 28% to 210% (mean = 105%, SD = 55.8%). CONCLUSION AND DISCUSSION: All studies showed that walking exercises improved walking distance in patients with intermittent claudication. Further research is needed to determine the optimal exercise program, the effect of adherence to the treatment protocol, and the duration of the effects following a formal exercise program.

Exercise Therapy↗

PubMed1996

Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse.

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21(sp) (mouse homolog of Rad21, S. pombe) and hHR21(sp) (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21sp mRNA was cell cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21sp transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21sp resided on chromosome 15D3, whereas hHR21sp localized to the syntenic 8q24 region. Elevated expression of mHR21sp in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes.

Amino Acid Sequence↗

PubMed1996

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

Amino Acid Sequence↗

PubMed1996

Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs.

Amino Acid Sequence↗

PubMed1996

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

Chromosome Inversion↗

PubMed1995