mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Interaction of Co,Mn, Mg and Al with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T_m values. Mn enhanced T_m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T_m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.     

Synthesis, DNA binding, and oxidative cleavage studies of Fe(II) and Co(III) complexes containing bioactive ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF(6))(2) (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L(1)L(2))] (PF(6))(3) (2) (where L(1)L(2) = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K(b) has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10(5) M(-1) for (1) and 4.8 × 10(5) M(-1) for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.     

Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.     

Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge

The present study reports two bacteria, designated 87I and 112A, which were isolated from soil and activated sludge samples from Hyderabad, India, and that are capable of producing poly-3-hydroxybutyrate (PHB). Based on phenotypical features and genotypic investigations, these microorganisms were identified as Bacillus spp. Their optimal growth occurred between 28°C and 30°C and pH 7. Bacillus sp. 87I yielded a maximum of 70.04% dry cell weight (DCW) PHB in medium containing glucose as carbon source, followed by 55.5% DCW PHB in lactose-containing medium, whereas Bacillus sp. 112A produced a maximum of 67.73% PHB from glucose, 58.5% PHB from sucrose, followed by 50.5% PHB from starch as carbon substrates. The viscosity average molecular mass (M _v) of the polymers from Bacillus sp. 87I was 513 kDa and from Bacillus sp. 112A was 521 kDa. All the properties of the biopolymers produced by the two strains 87I and 112A were characterized.     

Smoking related risk involved in individuals carrying genetic variants of CYP1A1 gene in head and neck cancer

Background: CYP1A1 is one of the commonest genes which had been widely investigated to find the risk of various malignancies in different ethnic groups. The polymorphism in these genes with a combination of environmental exposure has been hypothesized to confer a differential risk of cancer for individuals carrying these genetic variants. Based on this model, individuals with higher CYP1A1 activity would be at increased risk of cancer when exposed to high levels of smoke components. The proposed mechanism involves cytochrome P450 1A1 (CYP1A1), a gene that is inducible by xenobiotics to produce genetic susceptibility for malignancies. Patients and procedures: We performed a case–control study in 205 cases with histopathologically confirmed squamous cell carcinoma of head and neck and reported habits of bidi or cigarettes smoking and 245 similar controls to investigate the role of CYP1A1 polymorphisms in the risk of head and neck cancers especially among smokers of Hyderabad Indian population. Venous blood samples (5 ml) were collected from patients and control groups; genomic DNA was extracted and used for polymerase chain reaction (PCR) to determine the genotypes. RFLP assays were designed to detect each of the variant CYP1A1 alleles. Results and discussion: CYP1A1m1/m1 genotype (OR = 8.12, 95% CI: 3.27–21.30) and CYP1A1w1/m1 showed elevated risk when compared with CYP1A1w1/w1. Similarly CYP1A1w2/m2 (OR = 1.58, 95% CI: 0.94–2.67) and CYP1A1m2/m2 (OR = 6.31, 95% CI: 2.74–18.69) genotypes also showed elevated risk when compared with CYP1A1w2/w2 genotype. This data demonstrated that smoking was a risk factor for head and neck cancers. The m2 mutations were in close linkage disequilibrium with the m1 mutations; 53% m1 mutants had the mutation in the m2 site. Conclusion: Those individuals carrying at least one CYP1A1 m1 or m2 variant allele were at a 2-fold elevated risk for head and neck cancer. Our data clearly demonstrates that CYP1A1 is an important determinant in susceptibility to tobacco-induced head and neck carcinogens and there is an association between genetic polymorphism in the CYP1A1 locus and elevated risk of the type of smoking among Indians. This appears to be a new and important prognostic and diagnostic marker for determining the risk of head and neck cancers genetically.     

Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.