The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1

A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.     

Downregulation of protein kinase C-α enhances intracellular survival of Mycobacteria: role of PknG

Background  

Intracellular trafficking of mycobacteria is comprehensively dependent on the unusual regulation of host proteins. Recently, we have reported that infection of macrophages by Mycobacterium tuberculosis H37Rv (Rv) selectively downregulates the expression of PKCα while infection by Mycobacterium smegmatis (MS) does not.     

Protective and survival efficacies of Rv0160c protein in murine model of Mycobacterium tuberculosis

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) multi-gene families code for approximately 10 % of the Mycobacterium tuberculosis (Mtb) genome. These proteins are thought to be virulence factors that participate in impounding the host immune responses. While some members have been studied, the functions of most PE/PPE proteins are yet to be explored. The studies presented here have specifically characterized the roles of one of the PE proteins of Mtb, Rv0160c (PE4), in mycobacterial persistence and in prophylactic efficacy. We have expressed Rv0160c in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that the protein improves the survival of mycobacteria in macrophages and in mice. The protein has also shown its effect under physiological stress of bacteria, as evidenced by elevated expression in acidic and in hypoxic conditions. In mice, the level of Rv0160c was noticeably high during the chronic stage of tuberculosis. The seroreactivity of the protein against different categories of tuberculosis patients revealed a strong B-cell humoral response in freshly infected pulmonary tuberculosis patients. In mice, it exhibited increased IL-2, TNF, and IL-6 production. The antigenic properties of the protein directed towards the protective efficacy against the Mtb challenge. All together, our findings have identified Rv0160c as an in vivo expressed immunodominant antigen which plays a crucial role in the pathogenesis of mycobacterial disease and could prove to be a good preventive antigen for tuberculosis.     

Synthesis,characterization and in vitro biological evaluation of some novel 1,3,5-triazine–Schiff base conjugates as potential antimycobacterial agents

A series of some novel 1,3,5-triazine–Schiff base conjugates (1–32) have been synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 4 (4-Methoxy-6-methyl-N-(3,4,5-trimethoxybenzylidene)-1,3,5-triazin-2-amine), 11 (4-Methoxy-6-methyl-N-(2-hydroxy-3-bromo-5-chloro-benzylidene)-1,3,5-triazin-2-amine) and 24 (4-Methoxy-6-methyl-N-(1-(2,5-dihydroxyphenyl)ethylidene)-1,3,5-triazin-2-amine) exhibited a significant activity at 3.125, 6.25 and 6.25 μg/mL, respectively, when compared with the antitubercular drugs such as ethambutol (3.125 μg/mL), pyrazinamide (6.25 μg/mL) and streptomycin (6.25 μg/mL) and it could be a potential starting point to develop new lead compounds in the fight against Mycobacterium tuberculosis H37Rv.     

Wood Anatomy and the Development of Interxylary Phloem of Ipomoea hederifolia Linn. (Convolvulaceae)

In Ipomoea hederifolia Linn., stems increase in thickness by forming successive rings of cambia. With the increase in stem diameter, the first ring of cambium also gives rise to thin-walled parenchymatous islands along with thick-walled xylem derivatives to its inner side. The size of these islands increases (both radially and tangentially) gradually with the increase in stem diameter. In pencil-thick stems, that is, before the differentiation of a second ring of cambium, some of the parenchyma cells within these islands differentiate into interxylary phloem. Although all successive cambia forms secondary phloem continuously, simultaneous development of interxylary phloem was observed in the innermost successive ring of xylem. In the mature stems, thick-walled parenchyma cells formed at the beginning of secondary growth underwent dedifferentiation and led to the formation of phloem derivatives. Structurally, sieve tube elements showed both simple sieve plates on transverse to slightly oblique end walls and compound sieve plates on the oblique end walls with poorly developed lateral sieve areas. Isolated or groups of two to three sieve elements were noticed in the rays of secondary phloem. They possessed simple sieve plates with distinct companion cells at their corners. The length of these elements was more or less similar to that of ray parenchyma cells but their diameter was slightly less. Similarly, in the secondary xylem, perforated ray cells were noticed in the innermost xylem ring. They were larger than the adjacent ray cells and possessed oval to circular simple perforation plates. The structures of interxylary phloem, perforated ray cells, and ray sieve elements are described in detail.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.