Differential gene expression in Giardia lamblia under oxidative stress: Significance in eukaryotic evolution

Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production.     

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC⁵⁰) without the lectin domain, and mutant VCC^D617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10⁷ m⁻¹. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC⁵⁰ was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β₁-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

Independent overexpression of OsRab16A and AtDREB1A exhibit enhanced drought tolerance in transgenic aromatic rice variety Pusa Sugandhi 2

Journal of Plant Biochemistry and Biotechnology - Rice (Oryza sativa L.), the staple food in South-East Asian countries, is a water-intensive crop so that its productivity is highly affected by...     

Intracellular mediators of potassium-induced aldosterone secretion

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in 3H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.     

Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.     

Short-term therapy of atherosclerosis with low dose indomethacin: an experimental study

The effects of low dose indomethacin therapy in primary prevention of diet-induced atherosclerosis of rhesus monkeys was studied. The parameters studied were serum cholesterol concentration, thromboxane A2 (T x B2), prostacyclin (6-keto-PGF1 alpha) in serum/plasma, and the extent and intensity of coronary atherosclerosis. Although indomethacin did not affect serum cholesterol, it reduced serum T x B2 significantly (P less than 0.01). Plasma 6-keto-PGF1 alpha was not restored to the pretreatment level. A significant protective role of the drug was noted as far as coronary atherosclerosis is concerned (P less than 0.01).     

Limited proteolysis reveals a structural difference in the globular head domains of dephosphorylated and phosphorylated Acanthamoeba myosin II.

Phosphorylation at three sites at the tip of the tail of myosin II from Acanthamoeba castellanii inactivates the actin-activated Mg(2+)-ATPase activity of filamentous myosin and the in vitro motility activity of both monomeric and filamentous myosin. To seek a structural explanation for these effects, we examined the susceptibilities of dephosphorylated and phosphorylated myosins II to endoproteinases. Endoproteinase Arg-C cleaved myosin II preferentially at two sites in the globular head, Lys-621 and Arg-638, producing an NH2-terminal fragment of about 67,000 Da and a COOH-terminal fragment of about 112,000 Da. Dephosphorylated monomers and filaments were cleaved about 3 times more rapidly than their phosphorylated counterparts principally because of a much greater rate of cleavage at Arg-638; the ratio of cleavage at Arg-638:Lys-621 was about 3 for dephosphorylated myosins and about 0.5 for phosphorylated myosins. These data demonstrate that phosphorylation at the tip of the tail of Acanthamoeba myosin II causes a conformational change in the globular head that contains the catalytic sites; therefore, this conformational change may be related to the different catalytic and motile activities of the dephosphorylated and phosphorylated enzymes.     

Release of acid hydrolases in spectrum of human leprosy.

Release of acid hydrolases by blood monocytes (BM) of leprosy patients both before and after 6 months of chemotherapy was measured fluorimetrically. Monocyte cultures were set up for spontaneous as well as zymosan dependent enzyme release measured after 2 hrs and 24 hrs of culture. In the untreated multibacillary group (BL/LL) a significantly higher (P < 0.001) release of both B-glucuronidase (BG) and N-acetyl glucosaminidase (NAG) was observed compared to the paucibacillary group (BT/TT) and healthy controls. On comparing the BT/TT group with controls a significant decrease (P < 0.001) in zymosan dependent NAG release was observed in the former group at 2 hrs culture. After 6 months of antileprosy therapy, a significant decrease (P < 0.05) in BG release was observed from BM of multibacillary patients, whereas NAG activity increased significantly (P < 0.05) in the paucibacillary group compared to the controls. The results of the present study suggest that non-oxidative metabolic status of BM vary within the leprosy spectrum.     

Hepatoprotective activity of Azadirachta indica leaves on paracetamol induced hepatic damage in rats.

Effect of A. indica leaf extract on serum enzyme levels (glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, acid phosphatase and alkaline phosphatase) elevated by paracetamol in rats was studied with a view to observe any possible hepatoprotective effect of this plant. It was interesting to observe that serum enzyme levels were much elevated in paracetamol induced animals than in those receiving a combination of paracetamol and lead extract. It is stipulated that the extract treated group was protected from hepatic cell damage caused by paracetamol induction. The findings were further confirmed by histopathological study of liver.