Sickle cell gene haplotypes in Relli and Thurpu Kapu populations of Andhra Pradesh

We performed polymerase chain reaction analysis of 8 restriction-site polymorphisms in the beta-globin gene cluster to define haplotypes and provide hematological profiles of Relli and Thurpu Kapu caste populations in Andhra Pradesh, India. In all sickle cell homozygous subjects, the clinical manifestation of the disease is benign with elevated fetal hemoglobin levels (3.9%-21.1%). Clinical symptoms in some of the sickle cell homozygous subjects include jaundice, leg ulcers, and splenomegaly. Molecular analysis of the sickle cell gene (HBB*S) reveals the presence of the ubiquitous Arab-Indian haplotype in both populations. We encountered, for the first time, a rare, atypical haplotype ((+)-------) in a sickle cell homozygous individual of the Thurpu Kapu population, presumably the result of gene conversion.     

Production of a novel indole ester from 2-aminobenzoate by Rhodobacter sphaeroides OU5

Culture supernatants of Rhodobacter sphaeroides OU5 grown in the presence of 2-aminobenzoate gave an orange-red color-reaction with Salpers reagent, suggesting the presence of an indole derivative. This production was light-dependent and inducible only with 2-aminobenzoate. Replacement of 2-aminobenzoate with other 2-substituted benzoates did not result in the formation of indole. Fumarate appeared to be the conjugating molecule with 2-aminobenzoate, resulting in the formation of an indole derivative. The purified indole derivative was orange-brown in color, with a yields 0.34 mM from 1 mM 2-aminobenzoate. Infrared analysis suggested an indole ester and ¹H NMR analysis indicated an indole carboxylate, esterified with a terpenoid alcohol. The indole ester has a mass of 441 with the molecular formula C₂₇H₃₉NO₄. The IUPAC name of the compound is (3 E,5 E)-14-hydroxy-3,7,11-trimethyl-3,5-tetradecadienyl 2-(hydroxymethyl)-1 H-indole-3-carboxylate; and the common name given to this compound is sphestrin.     

Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer cell growth by inhibiting ErbB-2 and ERK1

A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by performing in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore, the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal human fibroblasts suggesting that these compounds are tumor-specific.     

Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus

A new actinomycete strain designated as BT-408 producing polyketide antibiotic SBR-22 and showing antibacterial activity against methicillin resistant Staphylococcus aureus has been characterized and found to be a novel strain of Streptomyces psammoticus. Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been optimized. Glucose and ammonium nitrate were found to be best carbon and nitrogen sources respectively for growth and antibiotic production. Similarly initial medium pH of 7.2, incubation temperature of 30 degrees C and incubation time of 96 h were found to be optimal. Optimization of medium and cultural conditions resulted in 1.82-fold increase in antibiotic yield.     

Down-regulation of p53 by double-stranded RNA modulates the antiviral response

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G(1) arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G(1) arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.     

The SRY-1532 site of the human Y chromosome is subject to recurrent single nucleotide mutations

Haplotype determination based on three Y-linked polymorphic sites, 92R7 (C/T), SRY-1532 (A/G), and YAP (-/+), in 127 males belonging to three caste Hindu populations of South India (Vizag Brahmins, Peruru Brahmins, and Kammas) and 13 males belonging to a migrant group (the Siddis) showed the existence of all four haplotypes (CA-, CG-, TG-, and TA-) under the YAP- background. This finding suggests that the reverse mutation (G-->A) at the SRY-1532 site, described earlier in the literature, is present in South Indian populations as well. The YAP+ mutation was seen in only five Siddi individuals. Four of these were of the CG+ haplotype structure, but a novel haplotype (CA+) was found in one male. To explain the occurrence of the six haplotypes found within these three sites, a haplotype tree is constructed that introduces a new reverse mutation at the SRY-1532 site (G-->A), occurring under the CG+ background after the migrant Siddi population arrived in India.