Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.     

Bayesian Total Internal Reflection Fluorescence Correlation Spectroscopy Reveals hIAPP-Induced Plasma Membrane Domain Organization in Live Cells

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.     

Involvement of thermoplasmaquinone-7 in transplasma membrane electron transport of Entamoeba histolytica trophozoites: a key molecule for future rational chemotherapeutic drug designing

The quinone composition of the transplasma membrane electron transport chain of parasitic protozoa Entamoeba histolytica was investigated. Purification of quinone from the plasma membrane of E. histolytica and its subsequent structural elucidation revealed the structure of the quinone as a methylmenaquinone-7 (thermoplasmaquinone-7), a napthoquinone. Membrane bound thermoplasmaquinone-7 can be destroyed by UV irradiation with a concomitant loss of plasma membrane electron transport activity. The abilities of different quinones to restore transplasma membrane electron transport activity in UV irradiated trophozoites were compared. The lost activity was recovered completely by the addition of thermoplasmaquinone-7, but ubiquinones are unable to restore the same. These findings clearly indicate that thermoplasmaquinone-7 acts as a lipid shuttle in the plasma membrane of the parasite to mediate electron transfer between cytosolic reductant and non permeable electron acceptors. This thermoplasmaquinone-7 differs from that of the mammalian host and can provide a novel target for future rational chemotherapeutic drug designing.     

Polymorphism in chemokine receptor genes and risk of acute myocardial infarction in North Indian population

Chemokines regulates the trafficking of leukocytes to the site of inflammation hence may be implicated in cardiac events. Currently no consistent effects have been revealed their role in acute myocardial infarction (MI). The aim of current study was to investigate the impact of human chemokine receptor genetic variants, CCR5-Δ32 insertion/deletion, CCR5-59029-A/G, CX3CR1-V249I and CX3CR1-T280 M on acute MI. 230 acute MI and 300 controls were examined. Patients carrying CCR5-Δ32 genotype were at three times higher risk of developing MI odds ratio (OR, 3.24, CI 1.127–9.356, P = 0.04). Significant association was found with risk of acute MI in recipients who possessed homozygous 59029-A allele (OR 1.47, CI 1.03–2.09, P = 0.03). While CX3CR1-I249 and M280 were found to be protective in MI patients with OR 0.46, CI 0.32–0.66, P < 0.0001 and OR 0.36, CI 0.24–0.55, P < 0.0001, respectively. It might be possible that risk of acute MI is associated with genetic variation in chemokine receptors, i.e., CCR5 and CX3CR1.     

Pyrethrin accumulation in elicited hairy root cultures of <Emphasis Type="Italic">Chrysanthemum cinerariaefolium</Emphasis>

The flowers of Pyrethrum (Chrysanthemum cinerariaefolium) are known to contain Pyrethrins that are naturally occurring potential insecticide. Hairy roots were induced from leaves of C. cinerariaefolium using Agrobacterium rhizogenes strain A4. The root clones were characterized in to four groups i.e. thick, unbranched (D2 and D5), thin, highly branched (D3), thick, branched (B2) and thick, highly branched (D1, D6). Six established hairy root clones showed the presence of pyrethrin and were selected for elicitation studies. Growth kinetics studies revealed highest growth index in hairy root clone D1 (592.0) followed by D6 and D3 on dry weight basis after 40 days of culture. The maximum pyrethrin content was found in the clone D3 (7.2 mg/g dw) which is comparable to the flowers obtained from the variety “Avadh”. Hairy root clone D2 (5.2 mg/g dw) and D6 (1.3 mg/g dw) contained pyrethrin but in less amount as compared to clone D3. The PCR analysis showed the presence of rol B and rol C genes in all the six hairy root clones while rol A was detected only in D2 clone. The methanolic extract of D3 clone showed antifungal activities against phytopathogenic fungal strains which were found maximum against Curvuleria andropogonis followed by Colletotrichum acutatum and Rhizoctonia solani. Hairy root clones D2, D3 and D6 were elicited with culture filtrate of endophytic fungus (Fusarium oxysporum) and bacteria (Bacillus subtilis). The culture filtrate (4.0?%v/v) of both the fungal and bacterial origin was found to be effective in enhancing the pyrethrin content in all the tested hairy root clones. Clone D3 showed maximum pyrethrin content on elicitation with F. oxysporum (9.7 mg/g dw) and B. subtilis (9.7 mg/g dw) culture filtrate, which is 32?% higher than the non elicited D3 hairy roots (7.2 mg/g dw). F. oxysporum also enhanced the hairy root growth resulting into the higher biomass yield of D3 (50?%) and D2 (76?%) in comparison to control non elicited hairy root clones of D3 and D2, respectively leading to higher pyrethrin yield.     

Survival adaptations of threeEuphorbia spp. in arid ecosystem

Three species ofEuphorbia (E. granulata, E. prostrata andE. hirta) exhibited both morphological and physiological adaptations in dry environmental conditions. High bound water, thick cuticle, dense hair covering and low stomatal index are some of the survival adaptations they developed under water stress conditions of the Indian arid zone.     

In vivo radioprotection studies of 3,3′-diselenodipropionic acid,a selenocystine derivative

3,3′-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to γ-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of γ-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45α and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body γ-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.