Capacity building of primary care physicians of the tea garden hospitals in Dibrugarh, Assam: A demonstration project.

BACKGROUND: The three most commonly occurring cancers in India are those of the breast, uterine cervix, and lip or oral cavity, together accounting for approximately 34% of all cancers. All the three cancers are amenable to prevention, early detection, and treatment through which the morbidity and mortality due to these cancers can be reduced. This pilot study was conducted to assess the operational feasibility of the national cancer screening guidelines. METHOD: This study was conducted in the Dibrugarh district of Assam in seven tea garden hospitals which serve as the primary health centers for the tea estate population in the Northeast region of India. The study intervention was a three-day training package designed to train primary care physicians in population-based screening for oral, breast, and cervical cancers. Knowledge evaluation and skill assessment were performed with a validated questionnaire and checklist, respectively. RESULTS: Pre and posttraining knowledge assessment showed significant gain in the knowledge levels of the participants in all topics. The greatest knowledge increase was seen in breast cancer (96.3%), followed by cervical cancer (57.5%), oral cancer (35.5%) and general cancer-related information (16.7%). The skill assessment done for each participant individually at the end of the training indicated a need for retraining all participants in breast cancer screening. CONCLUSION: The learnings from this study will be of great help in scaling up the capacity building programme for cancer screening when the nation-wide population-based cancer screening programme will be rolled out in the country.

Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene.

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from -35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (-35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the -10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the -10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.

Risk factors for cancer nasopharynx: a case-control study from Nagaland, India.

BACKGROUND: A high incidence of nasopharyngeal carcinoma has been reported from Nagaland, though it is considered to be a rare neoplasm in India. No case-control study to identify the risk factors of cancer nasopharynx has been conducted in this region. This study was undertaken to identify dietary and environmental risk factors for nasopharyngeal carcinoma relevant to this region. METHODS: A matched case-control study using neighbourhood controls was conducted. For each of the 47 cases identified, 2 apparently healthy neighbourhood controls were matched for age, sex and ethnicity. All information on dietary, environmental, social and demographic factors was collected. Univariate and multivariate logistic regression analysis using maximum likelihood method was used to analyse data. RESULTS: Consumption of smoked meat was found to be the risk factor for nasopharyngeal carcinoma (adjusted odds ratio = 10.8; 95% CI 3.0-39.0). History of using herbal nasal medicine was also found to be associated with nasopharyngeal carcinoma (OR = 21.9, CI = 6.8-71.4). However, exposure to a smoky atmosphere, betel-nut chewing, use of smokeless tobacco products, smoking and drinking habits were not found to be associated with nasopharyngeal carcinoma. CONCLUSION: This study reveals an association of nasopharyngeal carcinoma with consumption of smoked meat in Nagaland. The use of herbal nasal medicine seems to be an additional risk factor for nasopharyngeal carcinoma in Nagaland and needs further assessment.

Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I.

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. A negative cis-acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.

An upstream element containing an ETS binding site is crucial for transcription of the human presenilin-1 gene.

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from -118 to +178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5' deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between -22 and -6. A mutation altering only two nucleotides of the ETS consensus sequence present at -12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from -14 to -6 and surrounds the ETS motif present at -12. Thus, a crucial ETS element is present at -12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the -12 motif. Several Sp1 binding motifs at positions -70, -55, and +20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5' and 3' deletions indicated the presence of positive promoter elements between -96 and -35 as well as between +6 and +42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3' deletions from +178 to +107 decreased promoter activity by 80%. However, further deletion to +42 increased promoter activity by 3-4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-1 (XAP-1).

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. The distal element (-128 to -85) appears to be liver specific because it shows positive activity in HepG2 cells and negative activity in HeLa cells. ApoB gene regulatory factor-2 (BRF-2) interacts with the sequence (-104 to -85). BRF-2 has been purified from rat liver nuclear extract, and its molecular weight has been determined to be approximately 120 kDa [Zhuang et al. (1992) Mol. Cell. Biol. 12, 3183-3191]. In this paper we report the isolation of two isoforms of BRF-2 by further purification using high-performance liquid chromatography. Both isoforms produced a single approximately 120-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis detected by silver stain. The amino acid sequences of two tryptic peptides derived from HPLC-purified heavier BRF-2 isoform were determined to be YLAIAPPIIK and ALYYLQIHPQELR. These two peptides were found to share 100% sequence homology with human hepatitis B virus X associated protein-1 (XAP-1) and monkey UV-damaged DNA-binding protein (UV-DDB). Anti-peptide antisera raised against two synthetic peptides of XAP-1 recognized a approximately 120-kDa polypeptide band in both BRF-2 isoforms in a western blot analysis. By using apoB promoter fragments containing various internal deletions and a substitution mutation as templates for gel mobility shift assays, we identified the region between -104 and -85 as crucial for binding by the high-molecular weight form. In contrast, the lower molecular weight isoform bound to all apoB mutants tested. Anti-peptide 2 antiserum directed against XAP-1 was found to inhibit in vitro transcription of the apoB gene in rat liver nuclear extracts by 50%. These results suggest that BRF-2 and XAP-1 are structurally and immunologically highly related trans-activators of the apoB gene. We propose that BRF-2 exists both as a monomer (BRF-2M) and as a homooligomer. probably a homodimer (BRF-2D), in solution; oligomerization appears to be an essential step for imparting sequence-specificity to BRF-2 protein and thereby facilitating its role as a trans-activator of the apoB gene.

Molecular characterization of the Azotobacter vinelandii recF gene.

The recF gene from Azotobacter vinelandii (Av) has been cloned by complementation in an Escherichia coli (Ec) recF mutant. The sequence of 1568 bp has been determined and analyzed. It showed an open reading frame of 1092 nt coding for a 364-amino-acid (aa) polypeptide. The comparison of the deduced aa sequence of the recF of Av with those of other bacteria has elicited the presence of the four conserved domains thought to be essential for RecF function. A transcriptional fusion of a DNA fragment containing the promoter sequence of recF with the lacZ gene of Ec was constructed and 3-4-fold enhancement of promoter activity was observed upon UV induction.

Isolation and characterization of a locus from Azospirillum brasilense Sp7 that complements the tumorigenic defect of Agrobacterium tumefaciens chvB mutant.

The chromosomal virulence gene chvB of Agrobacterium tumefaciens is required for pathogenesis. A DNA fragment from the chvB locus can hybridize to DNA from Azospirillum brasilense Sp7. This DNA fragment could restore the tumorigenic activity of the chvB mutant strain A. tumefaciens A1011 towards leaf disks of Nicotiana tabacum. An NH2-terminal open reading frame, 480 codons long, was most likely responsible for the restoration of the tumorigenic activity. The A. brasilense sequence showed good homology with the NH2-terminal region of the ndvB gene of Rhizobium meliloti.

The Azotobacter vinelandii nifL-like gene: nucleotide sequence analysis and regulation of expression.

The nucleotide sequence of the Azotobacter vinelandii nifL-like gene (Av-nifL) was determined. The 1.9 kb sequence shows an open reading frame (ORF) of 1577 bp which encodes a polypeptide of 519 amino acids, with a calculated molecular weight of 57,793. Av-nifL has about 50% homology with the Klebsiella pneumoniae nifL gene (Kp-nifL) at the nucleotide level and a little more than 52% homology at the amino acid level. The N-terminal regions show more homology than the C-terminal regions. As is the case in K. pneumoniae, Av-nifL is located just upstream of the A. vinelandii nifA gene (Av-nifA) and both genes constitute an operon. The expression of Av-nifL, however, seems to be independent of NtrA and NtrC. Furthermore, Av-nifL expression is not autogenously regulated by NifA, unlike the case in K. pneumoniae. The expression of an Av-nifL::lacZ fusion in A. vinelandii is inhibited by novobiocin and coumermycin A, which are inhibitors of DNA gyrase.

Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions.

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.

Extractive spectrophotometric determination of trace amounts of iron(III) with benzyltriethylammonium chloride.

The ion-association complex formed between a thiocyanato-iron(III) ion and a benzyltriethylammonium ion is extracted into 1,2-dichloroethane, and its absorbance at 476 nm is used for determination of the iron. Beer's law is obeyed up to about 4 mug/ml iron concentration in the final solution. The molar absorptivity is 2.79 x 10(4) l.mole(-1).cm(-1).

Structure and nucleotide sequence of the heavy chain gene of HLA-DR.

We have used a 175-nucleotide-long primer extension product corresponding to the 5' end of HLA-DR alpha-chain mRNA to isolate a genomic clone from a human DNA library. The entire HLA-DR alpha gene is contained in two contiguous EcoRI fragments spanning about 7.5 kilobases (kb); most of the sequence has been determined. The 5' end of the gene is contained in a 4.4-kb fragment, and the coding segments and the 3' untranslated region are contained in a 3.1-kb fragment. The gene is split into five exons. The 5' untranslated region, the leader peptide, and the first two NH2-terminal amino acids are fused into the first exon. Exons 2 and 3 represent two extracellular coding domains of mature p34. The transmembrane domain, cytoplasmic domain, and part of the 3' untranslated region are merged into a fourth exon. The rest of the 3' untranslated region is in exon 5. The predicted amino acid sequence of mature p34, as deduced from its gene structure, has 229 residues and reveals a single potential disulfide loop (between cysteine residues 107 and 163) as well as a 22-amino acid residue membrane integrated segment (residues 193-214). Fifteen amino acids (residues 215-229) reside on the cytoplasmic side of the plasma membrane. There is considerable amino acid sequence homology between the second external domains of p34 and p29, as well as the immunoglobulin-like third domain of HLA-B7, and beta 2-microglobulin and the homologous constant region domains of the light and heavy chains of immunoglobulins.

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates.

Reticulocyte lysates contain a latent form of eukaryotic peptide chain initiation factor 2 (eIF-2) kinase (dsI) which becomes activated in the presence of double-stranded RNA and ATP and inhibits protein synthesis. The latent form of dsI has been partially purified from reticulocyte ribosomal salt wash. The purified dsI has been activated by incubation in the presence of poly(rI).poly(rC) and [gamma 32P]ATP and the activated dsI has been further purified to near homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified [32P]dsI shows an intensely staining 67,000-dalton polypeptide band which corresponds to a single 67,000-dalton radioactive band. During Sephadex (G-200) gel filtration, both the latent form of dsI and the activated dsI elute similarly with a peak corresponding to a molecular weight of 67,000. Purified dsI phosphorylates the 38,000-dalton subunit of eIF-2 and, under conditions of eIF-2 phosphorylation, dsI strongly inhibits AUG-dependent Met-tRNAf binding to 40 S ribosomes. Also, in partial reactions, eIF-2 alpha (P) formed by phosphorylation of eIF-2 using dsI and ATP, is not recognized by two eIF-2 ancillary factors, Co-eIF-2B and Co-eIF-2C. These results are similar to those reported previously for the heme-regulated eIF-2 kinase (Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidar, P., Das H. K., Yaghmai, B., Palmieri, S., and Gupta, N. K. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,5076-5079) and suggest that dsI, like the heme-regulated eIF-2 kinase phosphorylates eIF-2 and eIF-2 alpha (P) thus formed, in both cases, is not recognized by Co-eIF-2B and Co-eIF-2C, and is inactive in some step(s) of Met-tRNAf.40 S initiation complex formation.

Amino acid starvation in Escherichia coli K-12: characteristics of the translation process.

Some characteristics of the translation process during amino acid starvation in Escherichia coli have been examined. Once starvation has been established, premature termination of polypeptides is negligible and complete proteins are formed. There is some preference for the synthesis of shorter proteins. The number of ribosomes involved in protein synthesis appears to decline to about half during amino acid-starvation. The assembly time of proteins during amino acid starvation is increased to only about fourfold, though protein synthesis maintained by turnover is reduced to 10%. To explain these observations, a model has been proposed for the course of events that possibly take place from the onset of starvation.