Induction of nitric oxide production by natural killer cells: its role in tumor cell death.

It has been recognized that natural killer (NK) cells destroy AK-5 tumor cells, largely by cytolysis and apoptosis. The objective of this study was to elucidate the existence and the role of nitric oxide (NO) during this killing. The target cell killing ability of NK cells was associated with an increased production of NO with higher expression of inducible nitric oxide synthase. In part, the production of NO was confirmed by significant increase in cell lysis in the presence of l-arginine and attenuation of cell lysis, DNA fragmentation, and apoptosis by N(omega)-nitro-l-arginine methyl ester (L-NAME). An increased oxidation of intracellularly trapped dichlorofluorescein was observed in NK cells, which was effectively prevented by L-NAME. Exposure of AK-5 cells to chemically generated NO also induced DNA fragmentation in AK-5 cells. Further evidence for the involvement of NO in apoptosis was provided by the inhibition of specific cleavage of PARP and activation of CPP32 by L-NAME. Increased production of NO with simultaneous enhancement of the cytotoxic activity of NK cells from sc tumor-transplanted animals has been implicated in tumor regression when compared to the ip tumor-bearing animals. Overall, these observations suggest an important role for NO during NK cell-mediated apoptosis and lysis of AK-5 cells.     

Lead sorption by living biomass of Chroococcus multicoloratus and Oscillatoria trichoides: kinetics and equilibrium studies

We report here the ability of two freshwater living cyanobacteria, Chroococcus multicoloratus Wood and Oscillatoria trichoides Szafer, to remove lead (Pb²⁺) from aqueous solutions in batch system, wherein the effect of Pb²⁺ on the growth rate of both the cyanobacteria was evaluated. The influence of sorption time, initial pH, initial Pb²⁺ ion concentration, culture density and the biosorption equilibrium kinetics was examined. Biosorption capacity was found to be maximum between a pH of 5 and 5.14, on the second day of exposure and at an initial concentration of 80 and 60 mg L^?1 for C. multicoloratus and O. trichoides, respectively. An initial concentration in the range of 10–120 mg L^?1 significantly decreased the growth and efficiency of Pb²⁺ removal. The maximum sorptive capacity (q _max) obtained from the Langmuir isotherm for C. multicoloratus and O. trichoides was 178.57 and 106.38 mg g^?1, respectively. The pseudo-second-order model was found to correlate well with the experimental data. Metal recovery of 70–77 % was obtained with HCl as a desorbing agent. The results of Fourier transform infrared spectroscopy indicated the participation of hydroxyl, carboxyl and amino groups in the biosorption process. Based on our observations, we suggest that both species appear to be potential viable biosorbents for mildly acidic water contaminated with Pb²⁺.     

Biocontrol of root-rot disease of Coleus forskohlii and Coleus amboinicus by using plant extracts as antifungal agents

Different plant extracts were screened for their potential antifungal activity against Fusarium chlamydosporum causing root rot of Coleus amboinicus and Coleus forskohlii; the aqueous and 50% ethanol extract of Annona squamosa, Azadircta indica, Eucalyptus Spp., Ocimum sanctum, Lawsonia inermis, Allium schoenoprasum, Cinnamomum verum Zingiber officinale, Piper nigrum, Calendula officinalis species were found to be effective. Both aqueous and 50% ethanol extract of the aforementioned plants showed a significant inhibition.     

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

Single‐domain antibodies (sdAbs), the variable domains of camelid heavy chain‐only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti‐methotrexate, anti‐triclocarban and anti‐cortisol sdAbs revealed unexpected contributions of the non‐hypervariable “CDR4” loop, formed between β‐strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15‐acetyl‐deoxynivalenol (15‐AcDON), and to carbohydrates. We constructed and panned a phage‐displayed library in which CDR4 of the 15‐AcDON‐specific sdAb, NAT‐267, was extended and randomized. From this library, we identified one sdAb, MA‐232, bearing a 14‐residue insertion in CDR4 and showing improved binding to 15‐AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage‐displayed libraries in which the CDR4 and other regions of three hapten‐ or carbohydrate‐binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan‐binding specificities, we panned the library against four tumor‐associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten‐specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V_L domain.     

The sporadic occurrence of a group I intron-like element in the mtDNA rnl gene of Ophiostoma novo-ulmi subsp. americana

The presence of group I intron-like elements within the U7 region of the mtDNA large ribosomal subunit RNA gene (rnl) was investigated in strains of Ophiostoma novo-ulmi subsp. americana from Canada, Europe and Eurasia, and in selected strains of O. ips, O. minus, O. piceae, O. ulmi, and O. himal-ulmi. This insertion is of interest as it has been linked previously to the generation of plasmid-like mtDNA elements in diseased strains of O. novo-ulmi. Among 197 O. novo-ulmi subsp. americana strains tested, 61 contained a 1.6 kb insertion within the rnl-U7 region and DNA sequence analysis suggests the presence of a group I intron (IA1 type) that encodes a potential double motif LAGLIDADG homing endonuclease-like gene (HEG). Phylogenetic analysis of rnl-U7 intron encoded HEG-like elements supports the view that double motif HEGs originated from a duplication event of a single-motif HEG followed by a fusion event that combined the two copies into one open reading frame (ORF). The data also show that rnl-U7 intron encoded ORFs belong to a clade that includes ORFs inserted into different types of group I introns, e.g. IB, ID, IC3, IA1, present within a variety of different mtDNA genes, such as the small ribosomal subunit RNA gene (rns), apo-cytochrome b gene (cob), NADH dehydrogenase subunit 5 (nad5), cytochrome oxidase subunit 1 gene (coxI), and ATPase subunit 9 gene (atp9).

We also compared the occurrence of the rnl-U7 intron in our collection of 227 strains with the presence of the rnl-U11 group I intron and concluded that the U7 intron appears to be an optional element and the U11 intron is probably essential among the strains tested.     

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.

Methodology/Principal Findings

The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases ({"type":"entrez-protein","attrs":{"text":"ORF01222","term_id":"1178790230","term_text":"ORF01222"}}ORF01222, {"type":"entrez-protein","attrs":{"text":"ORF03896","term_id":"1178792974","term_text":"ORF03896"}}ORF03896, and {"type":"entrez-protein","attrs":{"text":"ORF01315","term_id":"1178790325","term_text":"ORF01315"}}ORF01315) exhibited the highest levels of up-regulation.

Conclusions/Significance

The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.