Phenotypic and genotypic characterization of B.t.LDC-391 strain that produce cytocidal proteins against human cancer cells

An indigenous Bacillus thuringiensis strain B.t.LDC-391 producing cytocidal proteins against human colon cancer cell line, HCT-116, was subjected to phenotypic and genotypic characterization to evaluate its relatedness to B.anthracis. The morphological features of this strain were meta-analyzed with data of other parasporin and insecticidal protein producing Bacillus thuringiensis strains. The conventional biochemical analysis and antibiotic sensitivity test proved it as an ampicillin resistant which is a salient feature, absent in B.anthracis Ames. PCR analysis showed the absence of cyt and parasporin related genes in the genome of B.t.LDC-391. But the strain was positive for cap gene. The sequencing and bio-informatic analysis of cap gene and 16S rDNA of B.t.LDC-391 placed it closer to B.thuringiensis and revealed significant divergence from that of any B.anthracis strain. However our strain lacked β- hemolysis on human erythrocytes which is a common feature of B.anthracis strains and parasporin producers.     

Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.

Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.     

Thermodynamic analysis of acetylation-dependent Pb1 bromodomain-histone H3 interactions

An acetyl-histone peptide library was used to determine the thermodynamic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains interact with histones by binding acetylated lysines. The bromodomain used in this study, BrD3, is derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin remodeling complex. Steady-state fluorescence anisotropy was used to examine the variations in specificity and affinity that drive molecular recognition. Temperature and salt concentration dependence studies demonstrate that the hydrophobic effect is the primary driving force, consistent with lysine acetylation being required for binding. An electrostatic effect was observed in only two complexes where the acetyl-lysine was adjacent to an arginine. The large change in heat capacity determined for the specific complex suggests that the dehydrated BrD3-histone interface forms a tightly bound, high-affinity complex with the target site. These explorations into the thermodynamic driving forces that confer acetylation site-dependent BrD3-histone interactions improve our understanding of how individual bromodomains work in isolation. Furthermore, this work will permit the development of hypotheses regarding how the native Pb1, and the broader class of bromodomain proteins, directs multisubunit chromatin remodeling complexes to specific acetyl-nucleosome sites in vivo.     

Evidence for diet partitioning among three coexisting native freshwater fishes in South Africa's Cape Fold Ecoregion

The partitioning of limited resources commonly explains how different species can coexist within the same ecological community. In this 2010 study, the diets of three coexisting freshwater fishes (Cape galaxias Galaxias zebratus, n = 27; Cape kurper Sandelia capensis, n = 60; Breede River redfin Pseudobarbus burchelli, n = 77) were characterised and compared in three headwater streams in South Africa's Cape Fold Ecoregion using gut contents and stable isotope analyses. These data were analysed to ascertain whether the three species exploit distinct trophic niches. Both approaches provided evidence that these species occupy different trophic niches, though with some overlap. However, dietary differences among sites were not consistent and were probably influenced by site-specific factors like resource availability. Pseudobarbus burchelli had a broader niche breadth at Tierkloof Stream than the other two species, but not at Waaihoek or Tierstel Streams. Our results also suggest that P. burchelli consumed a more omnivorous diet than do the other two species, whereas S. capensis occupied a higher trophic position than the other two species and consumed vertebrates. Our findings suggest that these species occupy non-equivalent feeding niches in Cape Fold Ecoregion headwater streams, and that diet partitioning might facilitate their coexistence in these systems.     

Conformational flexibilities in malformin A

The linked-atom-least-squares (LALS) technique has been applied to generate exactly cyclized and stereochemically satisfactory conformations of the cyclic pentapeptide, malformin A, which contains an intramolecular disulfide bridge across a D-Cys—D-Cys linkage. Consistent with theoretical, ir, and x-ray evidence from studies on analogs of the LKiysteinyl—L-cysteine disulfide, it is shown that the peptide bond across the S? S bridge in malformin A can retain a cis configuration. The two all-trans structures proposed earlier by A.E. Tonelli [(1978) Biopolymers 17, 1175–1179] from solution nmr data have also been analyzed. Both P-(χ^s ～ 90°) and M-(χ^s ～ ?90°)-type helicity of the S? S bridge are found to be accessible in both the trans and the cis models in which the respective conformations of the homodetic rings are essentially preserved. The details of six different conformational states and their relative energies have been evaluated and compared. Our findings, which suggest that a variety of conformational states are accessible to malformin A, are compatible with other published results from solution studies. On the basis of hydrogen-bonding interactions, a model is proposed to explain how malformin A might be inactivated specifically by L -Hyp, and not by L -Pro or other amino acids, as has been observed from in vivo investigations [Buckhout, T.J. & Curtis, R.W. (1976) Nature 260 , 435–436]     

Crystal and molecular structure of benzyloxycarbonyl-α-aminoisobutyryl-L-prolyl methylamide: The observation of an X2-Pro3 Type III β-Turn

The crystal and molecular structure of N-benzyloxycarbonyl-α-aminoisobutyryl-L -prolyl methylamide, the amino terminal dipeptide fragment of alamethicin, has been determined using direct methods. The compound crystallizes in the orthorhombic system with the space group P2₁2₁2₁. Cell dimensions are a = 7.705 Å, b = 11.365 Å, and c = 21.904 Å. The structure has been refined using conventional procedures to a final R factor of 0.054. The molecular structure possesses a 4 → 1 intramolecular N-H—O hydrogen bond formed between the CO group of the urethane moiety and the NH group of the methylamide function. The peptide backbone adopts the type III β-turn conformation, with ?₂ = ?51.0°, ψ₂ = ?39.7°, ?₃ = ?65.0°, ψ₃ = ?25.4°. An unusual feature is the occurrence of the proline residue at position 3 of the β-turn. The observed structure supports the view that Aib residues initiate the formation of type III β-turn conformations. The pyrrolidine ring is puckered in C^γ-exo fashion.     

Transfer and Expression of a Multiple Antibiotic Resistance Plasmid in Marine Bacteria

Conjugal transfer of a multiresistance plasmid from Pseudomonas fluorescens to halophilic and halotolerant bacteria was studied under in vitro and in situ conditions. Mating conducted in broth as well as on plates yielded a plasmid transfer frequency of as high as 10⁻³. Among these two, plate mating facilitated conjugal transfer of plasmid, because the cell-to-cell contact is more in plate mating. When P. fluorescens was incubated in seawater, the organism progressively lost its colony forming activity within 15 days. Microscopic examination revealed the presence of very short rods, indicating that the cells have become viable but nonculturable (VNC). Mating conducted in natural seawater without any added nutrients revealed that the conjugal transfer is influenced by the physical state of the donor and the recipients as well as the availability of nutrients. But a plasmid transfer frequency of 10⁻⁷ was obtained even after the donor cells have become VNC suggesting that the nonculturable state and nutrient deprived condition may not limit plasmid transfer. The results suggest that the terrestrial bacteria entering into the seawaters with antibiotic resistance plasmids may be responsible for the prevalence of resistance genes in the marine environment. Received: 4 May 1998 / Accepted: 18 June 1998     

Induction of defence-related enzymes in tomato (Solanum lycopersicum) plants treated with Bacillus subtilis CBR05 against Xanthomonas campestris pv. vesicatoria

Bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria is one of the most important destructive diseases of tomato in many parts of the agricultural world. Therefore, the present study aims to determine the effects of Bacillus subtilis CBR05 inoculation on bacterial spot disease severity and the induction of defence-related enzymes response in tomato. Tomato leaves were evaluated to determine the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO)) and the content of malondialdehyde (MDA). A reduction in bacterial spot severity was observed in plants inoculated with B. subtilis, compared with those of uninoculated controls. A significant increase in SOD, CAT, POD, and PPO activities was observed in plants treated with B. subtilis after 24?h inoculation compared with non-inoculated pathogen control and mock-inoculated controls. Moreover, the MDA content was induced by pathogen infection, and its amount in B. subtilis inoculated plants was significantly lower than that in pathogen control. Our results suggest that early increases in antioxidant enzymes and the reduction in MDA content with B. subtilis inoculation may play a pivotal role in mitigating oxidative stress, thereby induced systemic resistance against bacterial spot disease in tomato.