Effects of salt and denaturant on structure of the amino terminal alpha-helical segment of an antibacterial peptide dermaseptin and its binding to model membranes.

The amino terminal 1-18 domain of dermaseptin s is an important determinant of its structure as well as the antibacterial activity. A thorough investigation on the structure of the 18-residue peptide (D18) and its binding to model membranes in presence of salt and denaturant guanidinium chloride has been carried out. In presence of salt, there is an increase in the fraction of peptide molecules in helical conformation. In presence of the denaturant, D18 is unordered, but addition of the structure-promoting solvent trifluoroethanol results in a transition to the helical conformation. In presence of denaturant, the peptide is unordered, but binding to lipid vesicles is not abolished. Investigation of model membrane permeabilizing ability of the peptide in solutions containing various proportions of sodium chloride and guanidinium chloride indicates that vesicle permeabilization parallels extent of binding. The peptide thus binds to lipid vesicles in an unfolded state. Since the peptide has propensity to fold into a helical conformation, lipid induced transition to a helical structure occurs, followed by membrane permeabilization as a result of pore formation.     

Cathepsin B promotes both motility and invasiveness of oral carcinoma cells

We previously demonstrated that overexpression of cathepsin B (CB) protease in oral squamous cell carcinomas correlated positively with advanced tumor stage and poor histologic malignancy grade. Here we examined whether CB contributes to the invasiveness of oral carcinoma cells. For RNA-mediated inhibition, two ribozymes that target CB mRNA were designed and stably expressed in the oral squamous cell carcinoma cell line 1386Tu. Both ribozymes diminished expression of CB mRNA, protein, and activity, without affecting cathepsin D or beta-actin, as determined by quantitative real-time PCR, Western blots, and protease activity assays. Matrigel invasion assays showed that the invasiveness of the cells was significantly reduced by the expressed ribozymes and, surprisingly, the motilities of the ribozyme-transfected cells were also diminished. Our results document a direct role for CB in promoting oral cancer spread and invasion, and open the possibility of controlling oral carcinoma malignancy and metastasis by targeting CB with RNA inhibitor strategies.     

Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells

T cell tolerance is a critical element of tumor escape. However, the mechanism of tumor-associated T cell tolerance remains unresolved. Using an experimental system utilizing the adoptive transfer of transgenic T cells into naive recipients, we found that the population of Gr-1+ immature myeloid cells (ImC) from tumor-bearing mice was able to induce CD8+ T cell tolerance. These ImC accumulate in large numbers in spleens, lymph nodes, and tumor tissues of tumor-bearing mice and are comprised of precursors of myeloid cells. Neither ImC from control mice nor progeny of tumor-derived ImC, including tumor-derived CD11c+ dendritic cells, were able to render T cells nonresponsive. ImC are able to take up soluble protein in vivo, process it, and present antigenic epitopes on their surface and induce Ag-specific T cell anergy. Thus, this is a first demonstration that in tumor-bearing mice CD8+ T cell tolerance is induced primarily by ImC that may have direct implications for cancer immunotherapy.     

Pulmonary function and fuel use: A population survey

Background

In the backdrop of conflicting reports (some studies reported adverse outcomes of biomass fuel use whereas few studies reported absence of any association between adverse health effect and fuel use, may be due to presence of large number of confounding variables) on the respiratory health effects of biomass fuel use, this cross sectional survey was undertaken to understand the role of fuel use on pulmonary function.

Method

This study was conducted in a village of western India involving 369 randomly selected adult subjects (165 male and 204 female). All the subjects were interviewed and were subjected to pulmonary function test. Analysis of covariance was performed to compare the levels of different pulmonary function test parameters in relation to different fuel use taking care of the role of possible confounding factors.

Results

This study showed that biomass fuel use (especially wood) is an important factor for deterioration of pulmonary function (particularly in female). FEV₁ (p < .05), FEV₁ % (p < .01), PEFR (p < .05) and FEF_25–75 (p < .01) values were significantly lower in biomass fuel using females than nonusers. Comparison of only biomass fuel use vs. only LPG (Liquefied Petroleum Gas) use and only wood vs. only LPG use has showed that LPG is a safer fuel so far as deterioration of pulmonary function is concerned. This study observes some deterioration of pulmonary function in the male subjects also, who came from biomass fuel using families.

Conclusion

This study concluded that traditional biomass fuels like wood have adverse effects on pulmonary function.     

Tigerinins: novel antimicrobial peptides from the Indian frog Rana tigerina

Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenaline-stimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form beta-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.     

Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(R) electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6-1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 +/- 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 +/- 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.     

A synthetic strategy for ON-resin amino acid specific multiple fatty acid acylation of peptides

Covalent modification with fatty acids is observed in several proteins that play crucial roles in cellular physiology. In this paper, a convenient method for the generation of multiple fatty acylated synthetic peptides is described. Peptides were synthesized using solid phase procedures with fluorenylmethoxycarbonyl a-amino protected amino acids. Acetamidomethyl protected cysteines were employed. The thiol protecting group was selectively deprotected and acylation was carried out on the resin-bound peptides. The strategy described in this report is applicable to any peptide sequence.     

CD36 Contributes to Malaria Parasite-Induced Pro-Inflammatory Cytokine Production and NK and T Cell Activation by Dendritic Cells

The scavenger receptor CD36 plays important roles in malaria, including the sequestration of parasite-infected erythrocytes in microvascular capillaries, control of parasitemia through phagocytic clearance by macrophages, and immunity. Although the role of CD36 in the parasite sequestration and clearance has been extensively studied, how and to what extent CD36 contributes to malaria immunity remains poorly understood. In this study, to determine the role of CD36 in malaria immunity, we assessed the internalization of CD36-adherent and CD36-nonadherent Plasmodium falciparum-infected red blood cells (IRBCs) and production of pro-inflammatory cytokines by DCs, and the ability of DCs to activate NK, and T cells. Human DCs treated with anti-CD36 antibody and CD36 deficient murine DCs internalized lower levels of CD36-adherent IRBCs and produced significantly decreased levels of pro-inflammatory cytokines compared to untreated human DCs and wild type mouse DCs, respectively. Consistent with these results, wild type murine DCs internalized lower levels of CD36-nonadherent IRBCs and produced decreased levels of pro-inflammatory cytokines than wild type DCs treated with CD36-adherent IRBCs. Further, the cytokine production by NK and T cells activated by IRBC-internalized DCs was significantly dependent on CD36. Thus, our results demonstrate that CD36 contributes significantly to the uptake of IRBCs and pro-inflammatory cytokine responses by DCs, and the ability of DCs to activate NK and T cells to produce IFN-γ. Given that DCs respond to malaria parasites very early during infection and influence development of immunity, and that CD36 contributes substantially to the cytokine production by DCs, NK and T cells, our results suggest that CD36 plays an important role in immunity to malaria. Furthermore, since the contribution of CD36 is particularly evident at low doses of infected erythrocytes, the results imply that the effect of CD36 on malaria immunity is imprinted early during infection when parasite load is low.     

Probing the role of aromatic residues in the self-assembly of Aβ(16–22) in fluorinated alcohols and their aqueous mixtures

The Aβ(16–22) sequence KLVFFAE spans the hydrophobic core of the Aβ peptide and plays an important role in its self-assembly. Apart from forming amyloid fibrils, Aβ(16–22) can self-associate into highly ordered nanotubes and ribbon-like structures depending on the composition of solvent used for dissolution. The Aβ(16–22) sequence which has FF at the 19th and 20th positions would be a good model to investigate peptide self-assembly in the context of aromatic interactions. In this study, self-assembly of Aβ(16–22) and its aromatic analogs obtained by replacement of F19, F20 or both by Y or W was examined after dissolution in fluorinated alcohols and their aqueous mixtures in solvent cluster forming conditions. The results indicate that the presence of aromatic residues Y and W and their position in the sequence plays an important role in self-assembly. We observe the formation of amyloid fibrils and other self-assembled structures such as spheres, rings and beads. Our results indicate that 20% HFIP is more favourable for amyloid fibril formation as compared to 20% TFE, when F is replaced with Y or W. The dissolution of peptides in DMSO followed by evaporation of solvent and dissolution in water appears to greatly influence peptide conformation, morphology and cross-β content of self-assembled structures. Our study shows that positioning of aromatic residues F, Y and W have an important role in directing self-assembly of the peptides.     

Large-scale phosphosite quantification in tissues by a spike-in SILAC method

Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.