Associated factors with cervical pre-malignant lesions among the married fisher women community at Sadras, Tamil Nadu.

OBJECTIVE: To identify the associated factors of cervical pre-malignant lesions among the married fisher women residing in the coastal areas of Sadras, Tamil Nadu. METHODS: The study was conducted in five fishermen communities under Sadras, a coastal area in Tamil Nadu, India. Two hundred and fifty married fisher women residing in the area. Quantitative descriptive approach with a cross-sectional study design was used. Data were collected using a structured interview schedule for identifying the associated factors and Pap smear test was performed for identifying the pre-malignant cervical lesions among the married fisher women. Data were analyzed using descriptive and inferential statistics. RESULTS: Among 250 women, about six (2.4%) of them presented with pre-cancerous lesions such as atypical squamous cell of undifferentiated significance (ASCUS) - five (2%) and mild dysplasia one (0.4%). Majority of the women, about 178 (71.2%) women, had abnormal cervical findings. Statistical analysis showed a significant association of risk factors such as advanced age, lack of education, low socioeconomic status, using tobacco, multiparity, premarital sex, extramarital relationship, using cloth as sanitary napkin, etc. CONCLUSION: The study findings clearly show the increased vulnerable state of the fisher women for acquiring cervical cancer as they had many risk factors contributing to the same.

Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis.

Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.

Translation and processing of Bacillus amyloliquefaciens extracellular RNase.

Bacillus amyloliquefaciens extracellular RNase has been previously cloned and expressed in Bacillus subtilis. Site-specific mutagenesis experiments have identified codon -39 as the start site of translation. We have determined the primary signal peptide cleavage site of preprobarnase and propose a pathway for the conversion of probarnase to mature barnase.

Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences.

Secretion vectors based on the genes from Bacillus amyloliquefaciens P for alkaline protease (aprBamP) and neutral protease (nprBamP) were constructed. With both aprBamP and nprBamP, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. The new sites enabled us to fuse a heterologous gene to the expression and secretion elements. We used the protein A gene (spa) from Staphylococcus aureus as a heterologous gene. Bacillus subtilis cells carrying the resulting apr-spa or npr-spa gene fusions synthesized the fusion protein. B. subtilis cells were also capable of removing the signal peptide from the fusion protein, as indicated by the appearance of processed protein A into the growth medium. In addition, these gene fusions allowed us to identify the signal processing site of both the APR-SPA and NPR-SPA proteins.

Fusion of pro region of subtilisin to staphylococcal protein A and its secretion by Bacillus subtilis.

Subtilisin is synthesized as a preproenzyme in Bacillus subtilis. We fused that region of the subtilisin gene, (apr[BamP]), which encodes the signal sequence and pro region, to the mature gene sequence (spa) for a heterologous protein (staphylococcal protein A). B. subtilis cells harboring this gene fusion synthesized a fusion protein consisting of the signal and pro sequence of subtilisin fused to the protein A; the signal sequence was processed and a fusion protein (pro + protein A) was secreted into the growth medium.

Mapping of the glucose dehydrogenase gene in Bacillus subtilis.

A 4.0-kilobase DNA fragment containing the developmentally regulated gene for glucose dehydrogenase (gdh) from Bacillus subtilis was incorporated into the plasmid pGX345, which contains a marker conferring chloramphenicol resistance (cat). The resistance marker of the resulting integration vector was used to map the gdh gene on the B. subtilis chromosome. Using PBS1 transduction, the gene order was determined to be aroI cat (gdh) mtlB dal. The cat (gdh) marker was also cotransformable with mtlB. The genetic location of the gdh gene established by this indirect method was confirmed by the fact that the original phage lambda EF2, containing a 10-kilobase B. subtilis DNA fragment from which the 4-kilobase gdh region had been subcloned, also contained the mtlB gene.

Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein.

The genes for alkaline protease (apr[BamP]) and neutral protease (npr[BamP]) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis. The DNA sequences of apr[BamP] and npr[BamP] revealed, in each case, the presence of a large open reading frame. The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein. Based on DNA sequence, the start point of translation has been identified as amino acid residue - 107 for apr[BamP] and -221 for npr[BamP]. To demonstrate that the start point of translation of apr[BamP] in vivo is probably at codon -107, codon -103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis. Alkaline protease was produced from this ochre mutant derivative of apr[BamP] only when the host strain was Su+. The presence of a pro sequence may be common to all of the secreted proteases from bacilli.

Effect of purine and pyrimidine limitations on RNA synthesis in Bacillus subtilis.

The effects of varying the intracellular levels of GTP or UTP on the rate of RNA synthesis in Bacillus subtilis were studied. The levels of these nucleotides were manipulated by pyrimidine limitation in a pyr auxotroph, by purine limitation in a pur auxotroph, or by the addition of decoyinine , which specifically inhibits GMP synthesis. Decreased levels of UTP and GTP were accompanied by dramatically decreased synthesis and accumulation of stable RNAs (tRNA and rRNA), but mRNA synthesis was less affected. However, sporulation was initiated only when the intracellular level of GTP decreased.

Specificity and control of uptake of purines and other compounds in Bacillus subtilis.

Certain nucleotides control adaptation to changing nutrition or differentiation (sporulation) resulting from a general nutritional deficiency. To maintain the adaptation or differentiation process, once it has started, it may have been important for cells to evolve several independent and metabolically controllable systems enabling the uptake and metabolism of various nucleic acid bases or nucleosides. We have analyzed the cellular reactions with these compounds by measuring both their effect on growth and their uptake in appropriately chosen auxotrophic and uptake mutants. We have found one uptake system for guanine and hypoxanthine, another one for guanosine and inosine, and three other systems for adenine, adenosine, and uracil. The uptake systems of guanine-hypoxanthine and guanosine-inosine are inhibited by the stringent response to amino acid deprivation (increase of guanosine 5'-diphosphate-3'-diphosphate), but they do not depend on the concentration of GTP, which decreases during sporulation. In contrast, the uptake of Ura depends on the presence of GTP, regardless of whether a GTP decrease was produced by the stringent response or otherwise. This was the only uptake system whose decrease was always correlated with the onset of sporulation. The uptake of other compounds, e.g., alpha-methylglucoside and alpha-aminoisobutyric acid, decreased under some, but not all, sporulation conditions.

Chloramphenicol-inducible gene expression in Bacillus subtilis.

A cloned Bacillus pumilus cat gene expresses chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. The chloramphenicol inducibility trait was shown to be determined by a 234-bp region of the cloned DNA. Nucleotide sequence analysis of this 234-bp segment indicated that the cat ribosome-binding site occurs within a 40-bp region containing 14-bp terminal inverted repeat sequences. Transcription of this region into RNA should sequester the cat ribosome-binding site in a stable stem-loop conformation. Chloramphenicol-mediated destabilization of the stem-loop is suggested as the basis for the chloramphenicol inducibility phenotype.

Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase.

Bacillus subtilis glucose dehydrogenase (EC 1.1.1.47) has been purified from sporulating cell extract to apparent homogeneity (as determined by polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing). The enzyme purified as a single molecular species with no evidence for a multiple form of the enzyme. The B. subtilis glucose dehydrogenase has an apparent isoelectric point of 4.7-4.8 and an apparent Mr = 126,000 and is comprised of four subunits of Mr = 31,500 each. The glucose 2-deoxyglucose and glucosamine substrate specificity of the enzyme is similar to the substrate specificity for B. subtilis spore germination, suggesting that the spore glucose dehydrogenase may play some role in spore germination. The B. subtilis glucose dehydrogenase is extremely dependent on the presence of glycerol or other hydrophobic bond-stabilizing agents (or NAD) for retention of enzymatic activity, and the presence of glycerol (20% w/v) in the extraction and purification buffers was absolutely necessary for the successful purification of this enzyme.

Isolation of a developmental gene of Bacillus subtilis and its expression in Escherichia coli.

Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started. We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B. subtilis DNA. It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B. subtilis during sporulation. Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E. coli from a promoter within the B. subtilis genome.

Partial nucleotide limitation induces phosphodiesterase I and 5'-nucleotidase in Bacillus subtilis.

Changes in the specific activity of enzymes involved in the degradation of RNA and nucleotides were measured in Bacillus subtilis under conditions of guanine deprivation, which initiates sporulation, and uracil deprivation, which does not initiate sporulation. Whereas the specific activities of most of the enzymes studied increased by less than a factor of 3, those of 5'-mononucleotide-producing phosphodiesterase and 5'-nucleotidase increased at least eightfold under both deprivation conditions.

Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides.

When sporulation is initiated by nutrient limitation, e.g., at the end of growth, certain biochemical processes occur in sequence. To determine which of these processes occur, even when the cells sporulate in the presence of a rapidly metabolizable carbon source, we induced sporulation of Bacillus subtilis by deprivation of guanine nucleotides, in a synthetic medium containing excess glucose, ammonium ions, and phosphate. The deprivation was produced either by decoyinine addition to a standard strain or by guanosin limitation of a guanine auxotroph. At 1 h after the onset of this deprivation, an extensive turnover of proteins began whose appearance was chloramphenicol sensitive. At least one enzyme (aspartate transcarbamylase) lost 70% of its activity within 15 min, indicating its rapid destruction. Whereas the magnitude of the above two changes was similar to that observed during sporulation at the end of growth in nutrient sporulation medium, protease (intracellular and extracellular) increased to less than one-tenth of the specific activity in nutrient sporulation medium, and alkaline phosphatase increased to less than one-half. However, glucose dehydrogenase, an enzyme made only in forespores, increased to the same specific activity under both conditions, presumably because the forespore compartment is protected from media (e.g., glucose) influences by the double membrane (two bilayers with opposite polarity).

The role of manganese in growth and sporulation of Bacillus subtilis.

Phosphoglycerate phosphomutase of Bacillus subtilis, Bacillus cereus and Bacillus megaterium required Mn2+ as cofactor, whereas the wheat germ and rabbit liver enzymes did not. In the absence of Mn2+, B. subtilis did not sporulate in normal sporulation media but it did sporulate if the proper ratio of glucose or glycerol and malate was used. Decoyinine, an inhibitor of guanosine monophosphate synthesis, induced sporulation in the presence of excess glucose and malate to the same extent with and without Mn2+. Apparently, phosphoglycerate phosphomutase is the only strictly Mn2+-requiring enzyme needed for optimal sporulation in normal sporulation media.

On the possible mode of action of neurohormones on cholinesterase activity in the ventral nerve cord of scorpion, Heterometrus fulvipes.

It is shown that Km of ChE is not affected by the neurohormones but Vmax is increased and decreased in presence of acceleratory and inhibitory neurohormones respectively. Hence it is suggested that the neurohormones might modulate the enzyme activity be altering the maximal velocities (Vmax) rather than affecting the enzyme affinity (Km) towards the substrate.