Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.     

In vitro explant culture of mantle epithelium of freshwater pearl mussel

The in vitro culture of nacre secreting pallial mantle explants of freshwater pearl producing mussel, Lamellidens marginalis (Lamarck) included depuration of pearl mussels with different physical and chemical agents to eradicate various commensals, removal of pallial mantle ribbon, aseptic preparation of explants from the ribbon and transfer of those explants into tissue culture petri dishes. Special synthetic tissue culture media enriched with additives viz., inactivated calf fetal serum and antibiotics were poured into plates with explants. The culture plates were incubated at 30 degrees C in a CO2 incubator at 5%, CO2. The cultures could be maintained for 42-45 days without any contamination. After 12 hr epithelial like cells began to migrate out and formed a complete cell sheet surrounding the explant within 12-15 days. The epithelial cells in the culture indicated functional viability as subsequently after 38-40 days of culture, typical aragonitic 'nacre' crystals of CaCO3 could be observed throughout the culture plates.     

Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus

Background

Bacterial macrofibers twist as they grow, writhe, supercoil and wind up into plectonemic structures (helical forms the individual filaments of which cannot be taken apart without unwinding) that eventually carry loops at both of their ends. Terminal loops rotate about the axis of a fiber's shaft in contrary directions at increasing rate as the shaft elongates. Theory suggests that rotation rates should vary linearly along the length of a fiber ranging from maxima at the loop ends to zero at an intermediate point. Blocking rotation at one end of a fiber should lead to a single gradient: zero at the blocked end to maximum at the free end. We tested this conclusion by measuring directly the rotation at various distances along fiber length from the blocked end. The movement of supercoils over a solid surface was also measured in tethered macrofibers.

Results

Macrofibers that hung down from a floating wire inserted through a terminal loop grew vertically and produced small plectonemic structures by supercoiling along their length. Using these as markers for shaft rotation we observed a uniform gradient of initial rotation rates with slopes of 25.6°/min. mm. and 36.2°/min. mm. in two different fibers. Measurements of the distal tip rotation in a third fiber as a function of length showed increases proportional to increases in length with constant of proportionality 79.2 rad/mm. Another fiber tethered to the floor grew horizontally with a length-doubling time of 74 min, made contact periodically with the floor and supercoiled repeatedly. The supercoils moved over the floor toward the tether at approximately 0.06 mm/min, 4 times faster than the fiber growth rate. Over a period of 800 minutes the fiber grew to 23 mm in length and was entirely retracted back to the tether by a process involving 29 supercoils.

Conclusions

The rate at which growing bacterial macrofibers rotated about the axis of the fiber shaft measured at various locations along fibers in structures prevented from rotating at one end reveal that the rate varied linearly from zero at the blocked end to maximum at the distal end. The increasing number of twisting cells in growing fibers caused the distal end to continuously rotate faster. When the free end was intermittently prevented from rotating a torque developed which was relieved by supercoiling. On a solid surface the supercoils moved toward the end permanently blocked from rotating as a result of supercoil rolling over the surface and the formation of new supercoils that reduced fiber length between the initial supercoil and the wire tether. All of the motions are ramifications of cell growth with twist and the highly ordered multicellular state of macrofibers.     

Opercular Activity and Temporal Organization of Surfacing Behaviour in Indian Catfishes, Clarias batrachus and Heteropneustes fossilis

Opercular and surfacing activity were observed in the Indian catfish Clarias batrachus and Heteropneustes fossilis . In the series A experiments, the opercular activity was monitored under two experimental conditions, viz., (1) surfacing allowed and (2) surfacing prevented. A statistically significant elevation of the rate of opercular activity was observed when air breathing was prevented in both species. In addition, a significant prevention effect and a time of day dependence of that were noticed in both species. In the series B experiments, temporal patterns of surfacing and air-gulping activities were examined under an artificial LD 12:12 schedule at 2-h intervals over a period of 48 h. The inter air-gulping interval in minutes between two consecutive bouts was also recorded four times each day in both species. A significant 24-h rhythm was found for the rate of surfacing activity and length of the inter air-gulping interval in both species. However, the overall activity appears to be much higher in Heteropneustes fossilis as compared to Clarias batrachus .     

Enzymatic hydrolysis of malathion and other dithioate pesticides

Summary Constitutive, cell free enzyme extracts derived from two strains of Arthrobacter sp. SB 3 and SB 4 hydrolyzed malathion and exhibited K_m values of 1.3 and 2.0 mol/mg protein.min respectively. These two enzyme extracts had a broad pH optimum (6–9), a temperature optimum of 25 °C and 36 °C and were not strongly affected by high salt or solvent concentrations (up to 5%).     

The determination of n-alkanes in the cuticular wax of leaves of Ludwigia adscendens L

An n-hexane extract of fresh, mature leaves of Ludwigia adscendens, containing a thin layer of epicuticular waxes, has been analysed for the first time by TLC, IR and GC using standard hydrocarbons. The leaves contained 22 identified long chain (C15-C36) n-alkanes, accounting for 74.27% of the hydrocarbons present, and an unknown number of unidentified branched chain alkanes. The predominant n-alkane was C25 (11.02%), whilst C18 (7.62%), C20 (6.14%), C29 (5.36%) and C27 (5.29%) n-alkanes were moderately abundant: the C35 homologue was present only in minor amounts (0.22%).     

A novel class of dual-family immunophilins

Immunophilins are protein chaperones with peptidylprolyl isomerase activity that belong to one of two large families, the cyclosporin-binding cyclophilins (CyPs) and the FK506-binding proteins (FKBPs). Each family displays characteristic and conserved sequence features that differ between the two families. We report a novel group of dual-family immunophilins that contain both CyP and FKBP domains for which we propose the name FCBP (FK506- and cyclosporin-binding protein). The FCBP of Toxoplasma gondii, a protozoan parasite, contained N-terminal FKBP and C-terminal CyP domains joined by tetratricopeptide repeats. Structure-function analysis revealed that both domains were functional and exhibited family-specific drug sensitivity. The individual domains of FCBP inhibited calcineurin (protein phosphatase 2B) in the presence of the appropriate drugs. In binding studies, FCBP recruited calcineurin in the presence of FK506 and a putative target of rapamycin homolog in the presence of rapamycin. Two additional FCBP sequences in Flavobacterium and one in Treponema (spirochete) were also identified in which the CyP and FKBP domains were in the reverse order. T. gondii growth was inhibited by cyclosporin and FK506 in a moderately synergistic manner. The knockdown of FCBP by RNA interference revealed its essentiality for T. gondii growth. Clearly, the FCBPs are novel chaperones and potential targets of multiple immunosuppressant drugs.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.