Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma

4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.     

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.     

Differential responses of the photoperiodic clock in two passerine birds possessing a strongly self-sustained circadian system

To investigate whether the photoperiodic clocks of species possessing strongly self-sustaining circadian clocks share identical features, we compared the full response cycle (initiation and termination of the response) in body mass and testes of the non-migratory house sparrow (Passer domesticus) with that of the migratory redheaded bunting (Emberiza bruniceps) under Nanda-Hamner experiments. Birds were exposed to a 36 h day (L:D=6:30 h), controls exposed to a 24 h day (L:D=6:18 h), for a period of 31 weeks. By week 18 of L:D=6:18 h, there was a small increase in body mass among sparrows, but not among buntings, and the testes of bunting did not grow, while those of sparrow grew slightly. The response to L:D=6:30 h is of particular interest. There was a rapid gain and subsequent loss in the body mass of bunting, but not of sparrows. Further, both species underwent a testicular cycle as if they were exposed to long days, but the response of sparrows was slower and hence delayed the attainment of peak testicular size. Such a differential response to exotic light cycles between these two photosensitive species, despite their similar circadian oscillatory properties (strong self-sustainment), could suggest a species-specific adaptation of the endogenous clock involved in photoperiodic regulation of avian seasonality.     

Tracheal agenesis with broncho-esophageal fistula in VACTERL / TACRD association

Tracheal agenesis (TA) is an extremely rare malformation. We report here autopsy findings in a case of TA with bronchoesophageal fistula of Floyd type III. The other malformations present included laryngeal atresia, Right lung hypolobulation, ventricular septal defect in membranous portion, bilateral cystic renal dysplasia, spleninculus, Meckel''s diverticulum, and imperforate anus. The constellations of malformations present in our case have overlapping features with Vertebral anomalies, Anal atresia, Cardiovascular anomalies, Tracheo-esophageal fistula, Esophageal atresia, Renal anomalies, Limb anomalies and Tracheal atresia or laryngo tracheal atresia, Cardiac anomalies, Renal anomalies, Duodenal atresia association described previously in the literature.     

Otomycosis — a clinico-mycological study and efficacy of mercurochrome in its treatment

A total of 110 patients of symptomatic otomycosis was investigated, prospectively. Aural swabs were collected on 1st, 7th and 14th day and examined by direct microscopy and culture for fungi. Of these, 80 patients found to be having pure fungal infection, were taken up for mycological and therapeutic study. Fungi belonging to genus Aspergillus were isolated in 76 (95.0%) patients of which Aspergillus niger was the commonest isolate in 46 (57.5%), followed by A. flavus in 27 (33.7%), A. fumigatus in 3 (3.7%), Candida species in 3 (3.7%) and Mucor in 1 (1.2%). The patients were of all age groups but majority were between 21 and 30 years and the male-female ratio was equal. Of the total of 40 male patients, twenty-one were Sikhs using turban. Before developing the symptoms, forty five patients used oil, mixture of oil and garlic juice, antibiotics, steroids, antiseptics or wax solvent as ear drops. Only two patients were diabetic! No patient had fungal infection elsewhere in the body. The patients were called for regular follow-up for three weeks. In forty cases mercurochrome was applied as the antifungal agent after cleaning the external auditory canal, in twenty-three clotrimazole and in rest of the seventeen patients miconazole was used. On 7th day, only 11 (13.7%) patients grew different fungi in culture. They became symptom-free on 14th day and no fungal material could be seen on otoscopy, direct microscopy or culture. Mercurochrome was found to be most effective in these patients.     

Assessment of functional and genetic diversity of aerobic endospore forming Bacilli from rhizospheric soil of Phyllanthus amarus L.

Fifty two aerobic and endospore forming Bacilli (AEFB) strains were recovered from rhizospheric soil of Phyllanthus amarus. Morphological, biochemical and molecular characterization by 16S rDNA gene sequencing has shown that these bacterial strains belong to six different genera of AEFB i.e. Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, Terribacillus and Jeotgalibacillus. Analysis of their PGP activities has shown that 92.30 % strains produced indole acetic acid hormone, 86.53 % of the strains solubilized Phosphate and 44.23 % strains produced siderophore. Chitinase production activity was shown by 42.30 % of the strains and 21.15 % of the strains produced 1-amino cyclopropane-1-carboxylate (ACC) deaminase. 46.15 % of isolates have shown antagonistic activity against common fungal pathogen of the plant i.e. Corynespora cassiicola. Among all of the isolated strains B. Cereus JP44SK22 and JP44SK42 have shown all of the six plant growth promoting traits tested. B. megaterium strains (JP44SK18 and JP44SK35), Lysinibacillus sphaericus strains (JP44SK3 and JP44SK4) and Brevibacillus laterosporus strain JP44SK51 have also shown multiple PGP activities except ACC deaminase production activity. In the present study bacterial strain belonging to genera Jeotgalibacillus sp. JP44SK37 has been reported first time as a member of rhizospheric soil habitat and has also shown PGP activities. It can be concluded that Rhizosphere of P. amarus has harboured a good diversity of AEFB bacterial strains having a lot of biofertilizing and biocontrol abilities.     

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.