Aggregation and porin-like channel activity of a beta sheet peptide

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.     

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [³H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP) (500 μM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [³H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.     

Conversion of a porin-like peptide channel into a gramicidin-like channel by glycine to D-alanine substitutions

The beta-barrel and beta-helix formation, as in porins and gramicidin, respectively, represent two distinct mechanisms for ion channel formation by beta-sheet proteins in membranes. The design of beta-barrel proteins is difficult due to incomplete understanding of the basic principles of folding. The design of gramicidin-like beta-helix relies on an alternating pattern of L- and D-amino acid sequences. Recently we noticed that a short beta-sheet peptide (xSxG)(6), can form porin-like channels via self-association in membranes. Here, we proposed that glycine to D-alanine substitutions of the N-formyl-(xSxG)(6) would transform the porin-like channel into a gramicidin-like beta(12)-helical channel. The requirement of an N-formyl group for channel activity, impermeability to cations with a diameter >4 A, high monovalent cation selectivity, and the absence of either voltage gating or subconductance states upon D-alanine substitution support the idea of a gramicidin-like channel. Moreover, the circular dichroism spectrum in membranes is different, indicating a change in regular beta-sheet backbone structure. The conversion of a complex porin-like channel into a gramicidin-like channel provides a link between two different mechanisms of beta-sheet channel formation in membranes and emphasizes the importance of glycine and D-amino acid residues in protein folding and function and in the engineering of ion channels.     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

The saponin-rich fraction of a Gymnema sylvestre R. Br. aqueous leaf extract reduces cafeteria and high-fat diet-induced obesity

We examined the antiobesity effect of a saponin-rich fraction of a Gymnema sylvestre R. Br. aqueous leaf extract (SGE) using cafeteria and high-fat diet-induced obese rats for a period of eight weeks. SGE was orally administered at a dose of 100 mg/kg body weight once a day to the treatment group. It significantly decreased the body weight, food consumption, visceral organs weight, and the levels of triglycerides, total cholesterol, low-density lipoproteins, very low-density lipoproteins, atherogenic index, glucose, and increased the levels of high-density lipoproteins. There was no significant difference with respect to all parameters of the study in case of normal (N) diet and N diet + SGE rats. In vitro, SGE inhibited the pancreatic lipase activity. The present study gave clear evidence that the SGE has a significant antiobese action, supporting its use in traditional medicine, and can be used as a substitute for synthetic drugs.     

Pod surface exudates of wild relatives of pigeonpea influence the feeding preference of the pod borer, Helicoverpa armigera

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives of crops with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we studied the feeding behavior of pod borer, Helicoverpa armigera, which is the most damaging pest of pigeonpea, in relation to biochemical characteristics of the pod surface exudates in a diverse array of germplasm accessions belonging to 12 species of pigeonpea wild relatives. Feeding by H. armigera larvae was significantly lower on the unwashed or water-, methanol-, or hexane-washed pods of Canajus sericeus, C. scarabaeoides, Flemingia bracteata, F. stricta, and Rhynchosia aurea than those of C. acutifolius, C. albicans, C. cajanifolius, C. lineatus, D. ferruginea, P. scariosa, R. bracteata, and the cultivated pigeonpea, C. cajan genotypes, ICPL 87, and ICPL 332, although there were a few exceptions. The methanol-washed pods of wild relatives were less preferred for feeding by the H. armigera larvae than the unwashed pods, but the hexane-washed pods were preferred more than the unwashed pods. The results suggested that methanol extracted the phagostimulants from the pod surface, while hexane removed the antifeedants. The high-performance liquid chromatography (HPLC) finger printing of methanol and hexane pod surface extracts showed qualitative and quantitative differences in compounds present on the pod surface of different wild relatives of pigeonpea. Some of the peaks in HPLC profiles were associated with feeding preference of the third-instar larvae of H. armigera. There was considerable diversity in wild relatives of pigeonpea as revealed by principal component analysis based on HPLC fingerprints of pod surface extracts in methanol and hexane, and H. armigera feeding on the pods. Wild pigeonpea accessions with low amounts of phagostimulants and high amounts of antifeedants may be used for introgression of resistance genes into the cultivated pigeonpea to develop varieties with broad-based resistance to H. armigera. There is considerable diversity among the wild relatives of pigeonpea, and the accessions with resistance to pod borer. These can be used to broaden the basis and increase the levels of resistance to H. armigera.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Screening for probiotic properties of strains isolated from feces of various human groups

The present study searched for potential probiotic strains from various human fecal samples. A total of 67 aerobic and 38 anaerobic strains were isolated from 5 different categories of human feces. Systematic procedures were used to evaluate the probiotic properties of the isolated strains. These showed about 75-97% survivability in acidic and bile salt environments. Adhesion to intestinal cell line Caco-2 was also high. The isolates exhibited hydrophobic properties in hexadecane. The culture supernatants of these strains showed antagonistic effects against pathogens. The isolates were resistant to a simulated gastrointestinal environment in vitro. Of the 4 best isolates, MAbB4 (Staphylococcus succinus) and FIdM3 (Enterococcus fecium), were promising candidates for a potential probiotic. S. succinus was found to be a probiotic strain, which is the second such species reported to date in this particular genus. A substantial zone of inhibition was found against Salmonella spp., which adds further support to the suggestion that the probiotic strain could help prevent intestinal infection. This study suggested that the human flora itself is a potential source of probiotics.     

Prevalence of pulmonary tuberculosis - a baseline survey in central India

Background

The present study provides an estimate of the prevalence of bacteriologially positive pulmonary tuberculosis in Jabalpur, a district in central India.

Methodology/Principal Findings

A community based cross-sectional survey was undertaken in Jabalpur District of the central Indian state of Madhya Pradesh. A stratified cluster sampling design was adopted to select the sample. All eligible individuals were questioned for pulmonary symptoms suggestive of TB disease. Two sputum samples were collected from all eligible individuals and were examined by Ziehl-Neelsen smear microscopy and solid media culture methods. Of the 99,918 individuals eligible for screening, 95,071 (95.1%) individuals were screened. Of these, 7,916 (8.3%) were found to have symptoms and sputum was collected from 7,533 (95.2%) individuals. Overall prevalence of bacteriologically positive PTB was found to be 255.3 per 100,000 population (95% C.I: 195.3–315.4). Prevalence was significantly higher (p<0.001) amongst males (355.8; 95% C.I: 304.4–413.4) compared with females (109.0; 95% C.I: 81.2–143.3). Prevalence was also significantly higher in rural areas (348.9; 95% C.I: 292.6–412.8) as compared to the urban (153.9; 95% C.I: 123.2–190.1).

Conclusions/Significance

The TB situation in Jabalpur district, central India, is observed to be comparable to the TB situation at the national level (255.3 versus 249). There is however, a need to maintain and further strengthen TB control measures on a sustained and long term basis in the area to have a significant impact on the disease prevalence in the community.