Ca(2+)-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.     

Antidiabetic effect of Cogent db,a herbal drug in alloxan-induced diabetes mellitus

Cogent db, a compound herbal drug, was investigated for its possible antidiabetic effect in alloxan-induced diabetic rats. Oral administration of 0.15, 0.30 and 0.45 g/kg body wt. of the aqueous solution of Cogent db for 40 days exhibited a significant reduction in blood glucose, glycosylated haemoglobin and increased plasma insulin, total haemoglobin along with antihyperlipidemic effects in diabetic rats. The effective dose was found to be 0.45 g/kg body wt. It also prevents body weight loss in diabetic rats. An oral glucose tolerance test (OGTT) was also performed in experimental diabetic rats in which there was a significant improvement in glucose tolerance in rats treated with Cogent db. A comparison was made between the action of Cogent db and a known antidiabetic drug — glibenclamide (600 μg/kg body wt.). The antidiabetic effect of Cogent db was more effective than that observed with glibenclamide.     

Rapid population declines of Egyptian vulture (Neophron percnopterus) and red-headed vulture (Sarcogyps calvus) in India

Since the early 1990s, large and rapid population declines of three species of vulture ( Gyps spp.) endemic to south Asia have occurred on the Indian subcontinent and have led to these species being listed by IUCN as critically endangered. Evidence of rates of population decline, cause of death and toxicity is consistent with these declines being caused by poisoning of vultures through the ingestion of tissues from livestock treated with the anti-inflammatory drug diclofenac. In this paper, analysis of repeated surveys in and near protected areas widely spread across India shows that populations of two other vulture species, Egyptian vulture Neophron percnopterus and red-headed vulture Sarcogyps calvus , have also declined markedly and rapidly, but probably with a later onset than Gyps vultures in the same region. The declines continued at least up to 2003. It is recommended that these two species are considered for inclusion in the IUCN Red List and for urgent remedial conservation measures. Research is needed to determine whether or not the principal cause of these declines is diclofenac poisoning and to establish population trends in other scavenging birds in the Indian subcontinent.     

Assessment of post extraction complications in Indians

Extraction is one of the commonest procedures in dentistry. Therefore, it is of interest to evaluate the post extraction complications in patients undergoing extractions of permanent teeth. A total of 70 adult patients who had undergone dental extractions and presented with post -operative complications were included in the study and evaluated. Data collected was statistically analyzed using SPSS software and results obtained. Most of the patients with post extraction complications were in the age group of 31-40 years (21.6%), followed by 21-30 (20.2%) and 61-70 years (20.2%). Dry socket (39.19%) was the common post extraction complication in our study especially in the age group of 31-40 years. There was a statistically significant association between age of the patients and the post extraction complications (p<0.001). In our study, post extraction complications were commonly observed in age group of 31-40 years with a predilection for males. Dry socket was the most common post extraction complication. Age of the patient has a significant effect on post extraction complications. However, gender, smoking habits and systemic diseases have no influence on post extraction complications.     

In vitro cytotoxicity analysis of Zizyphus spina-christi stem bark extract on human cancer cell lines

Zizyphus spina-christi (Rhamnaceae family) is an edible plant used in folk medicine. Therefore, it is of interest to report the cytotoxic effects of Z. spina-christi bark crude extract on human cell lines. Crude ethanol extract of Z. spina-christi bark was fractionated with increasing polarity (diethyl ether, chloroform, ethyl acetate and butanol fractions). The fractions were examined for their cytotoxicity against human colon cancer (HCT-116 and CACO-2), cervical cancer (HeLa and HEp-2), lung carcinoma (A-549), hepatocellular carcinoma (HepG-2), breast cancer (MCF-7) and prostate cancer (PC-3) cell lines using viability assay. Diethyl ether fraction of Z. spina-christi showed the highest cytotoxic effects among the four extracts of Z. spina-christi. The IC50 of diethyl ether fraction was 7.14, 11.2, 11.6, 15.4, 39.8, 42.2, 84.2 and 153.8 µg/ml on HepG-2, A-549, CACO-2, HCT-116, MCF-7, PC-3, HeLa, and HEp-2 cell lines, respectively. Data shows that the diethyl ether fraction of Z. spina-christi showed effective cytotoxic effects in colon, lung and hepatocellular cancer cell lines.     

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3′ in the presence of Mg²⁺ as shown generating 3′ four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ββα-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD…D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.     

Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.     

T=1 capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T=3 and T=1 capsid assembly

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NDelta36) and 65 (CP-NDelta65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NDelta65 lacks the "beta-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NDelta36 mutant has the beta-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NDelta36 and CP-NDelta65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NDelta65, were mutated to asparagine (CP-NDelta65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.     

Hydrodynamic study of biogranules obtained from an anaerobic hybrid reactor

The bed expansion characteristics of a fluidized bed containing bacterial granules have been studied. These biogranules were obtained from an anaerobic hybrid reactor, which uses biogranules (without carrier particle) in fluidized condition. The settling velocity study of biogranules has shown that the drag coefficient of biogranule is greater than that of the rigid particle at the same Reynolds number. A new correlation based on this finding has been developed. The bed expansion study has demonstrated that a linear relationship exists between the natural logarithm of bed porosity and the natural logarithm of upflow superficial liquid velocity for the bed containing either a particular fraction of biogranule size or biogranules with wide size distribution. For a fluidized bed having a particular granule size, the bed porosity, and liquid superficial velocity could be related by the classic equation suggested by Richardson and Zaki (1954). The characteristic parameter of this correlation, the slope of the line n, has been related with Reynolds number. The intercept of the line gave a smaller value than the unhindered settling velocity of the particle. For fluidized bed having wide size distribution, the characteristic parameter n could not be related to Reynolds number. But the correlation suggested for single biogranule size has been found to predict n value with an average error of 2.3%.