Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.     

Dose- and duration-dependent alterations by tellurium on lipid levels: differential effects in cerebrum,cerebellum, and brain stem of mice

The effect of various doses of sodium tellurite (1/50 LD50=0.4 mg/kg, 1/25 LD50=0.8 mg/kg, and 1/10 LD50=2.0 mg/kg body weight orally) on the lipid levels (cholesterol, triglycerides, phospholipids, esterified fatty acids, gangliosides, and total lipids) in the cerebrum, cerebellum, and brainstem of male albino mice was studied after 7 and 15 d of treatment. Sodium tellurite (2.0 mg/kg body weight) for 7 d has an apparent effect on the depletion of cholesterol, triglycerides, phospholipids, esterified fatty acids, and total lipids. The cholesterol content was decreased significantly in the cerebrum, cerebellum, and brainstem after 7 d of treatment with a 2.0-mg/kg dose compared to the control. On the other hand, treatment for 15 d with doses of 0.4, 0.8, and 2.0 mg/kg body weight resulted in a significant and dose-dependent increment in cholesterol level in the cerebrum, cerebellum, and brainstem. The triglycerides content was decreased significantly in the cerebrum, cerebellum, and brainstem with the 2.0-mg/kg dose after 7 d of treatment. The doses of 0.4, 0.8, and 2.0 mg/kg orally for 15 d resulted in a significant and dose-dependent depletion of triglycerides in the cerebrum, cerebellum, and brainstem. All the doses of tellurium (0.4, 0.8, and 2.0 mg/kg) both for 7 and 15 d have depleted the level of phospholipids in varying degrees of significance in the cerebrum, cerebellum, and brainstem. However, the level of esterified fatty acids was decreased significantly with the 2.0-mg/kg dose of tellurium for 7 d but increased with the 0.4-mg/kg dose for 15 d in the cerebrum and cerebellum. The level of gangliosides was depleted in the cerebrum but elevated in the cerebellum and brainstem after receiving a 2.0-mg/kg dose of sodium tellurite for 7 d. The content of gangliosides was increased with doses of 0.4 and 0.8 mg/kg but decreased with 2.0 mg/kg for 15 d in the cerebrum, cerebellum, and brainstem. The total lipids content was depleted significantly and dose dependently after 7 and 15 d of treatment in the cerebrum, cerebellum, and brainstem. These results suggest that sodium tellurite affects the lipids content differentially in various parts of the mice brain.     

Investigation into the distribution of total,free, peptide-bound,protein-bound,soluble- and insoluble-collagen hydroxyproline in various bovine tissues

Collagen is a family of proteins which consists of several genetically distinct molecular species and is intimately involved in tissue organization, function, differentiation and development. The purpose of this study was to investigate the concentration of different hydroxyproline (Hyp) fractions viz., total, free, peptide-bound, protein-bound, soluble- and insoluble-collagen hydroxyproline (Hyp) in various bovine tissues. Results showed that liver had the highest concentration of free Hyp followed by kidney, brain, spleen, lungs, muscle and heart. Liver also had the highest concentration of peptide-bound collagen Hyp followed by kidney, heart, spleen, lungs, brain and muscle. The concentration of protein-bound collagen Hyp was highest in the liver, followed by kidney, spleen, lungs, muscle, brain and heart. Total Hyp was highest in the liver, followed by kidney, spleen, brain, heart, muscle and lungs. Liver also had significantly high concentration of collagen as compared to other tissues examined (P<0.001). Spleen had the significantly higher concentration of soluble-collagen Hyp when compared to other tissues (P<0.001). This was followed by heart, muscle, lungs, brain, kidney and liver. Heart had the highest concentration of insoluble-collagen Hyp followed by lungs, kidney, liver, muscle, spleen and brain. The variation among the insoluble-collagen Hyp concentration of heart and muscle, spleen and brain was significant (P<0.001). We speculate that these differences could be due to the variation in turn over of rate of collagen metabolism in this species.     

Study of alanine-73 and aspartate-9 of HLA-C locus in Saudi psoriasis patients, using sequence-specific primers (PCR-SSP)

Alanine at residue 73 (Ala-73) and aspartate at residue 9 (Asp-9) are characteristic to both Cw6 and Cw7 alleles of HLA-C gene and have been suggested as possible markers for psoriasis vulgaris (PsV). However, the results from various ethnic groups/populations are contradictory and inconclusive. In this study, an attempt has been made to examine the association between HLA-C (Ala-73 and Asp-9) and susceptibility to PsV among Saudi patients. Genomic DNA was extracted from 25 Saudi PsV patients and 75 control subjects. Polymerase chain reaction (PCR) was performed to amplify HLA-C sequences using earlier reported primers, C133P and C243PR. Sequence-specific primers were used to specifically detect nucleotide coding for Ala-73 and Asp-9 in all the subjects. The results showed significantly higher frequency of Asp-9 (84.0 % versus 61.3 %) in PsV patients as compared to controls (p < 0.05, 2-tailed Fisher's exact test). The frequencies of Ala-73 among PsV patients (92 %) and controls (88 %) did not differ significantly.     

Specific distribution of overexpressed aurora B kinase in interphase normal epithelial cells

Background  

It is known that aurora B, a chromosomal passenger protein responsible for the proper progression of mitosis and cytokinesis, is overexpressed throughout the cell cycle in cancer cells. Overexpression of aurora B produced multinuclearity and induced aggressive metastasis, suggesting that overexpressed aurora B has multiple functions in cancer development. However, the detailed dynamics and functions of overexpressed aurora B are poorly understood.     

The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine

Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.