Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis

The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain reduced secretion to the extracellular space. Many of these mutants were undetectable, but secretion of a subset of mutants with insertions in the N-terminal half of the toxin could be restored to wild type secretion levels if cultured in the presence of 0.1% Triton X-100. Secretion of two mutants with insertions at the extreme C terminus was partially restored when co-expressed with a minimal clone of EspP, a related autotransporter protein. Several well secreted mutants with insertions in the N-terminal third of the molecule reduced protease activity over 20-fold, suggesting that the protease domain is located within this N-terminal region of Pet. We have also identified two insertional mutants in the middle of the passenger domain that were proteolytic but no longer cytopathic; these mutants displayed decreased binding and internalization upon incubation with HEp-2 cells. Our data suggest the existence of separate functional domains mediating Pet proteolysis, secretion, and cell interaction.     

Origin binding activity of the Mycobacterial plasmid pAL5000 replication protein RepB is stimulated through interactions with host factors and coupled expression of repA

The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (ori) and two tandemly organized replication genes, repA and repB, the functions of which are not clearly known. It was observed that when the repA and repB genes were expressed in Escherichia coli, a strong ori binding activity was generated in the host cells. Inactivation of repB led to a complete loss of activity, whereas inactivation of repA had a partial effect, indicating that while repB plays an important role in the process, its activity is stimulated through coexpression of repA. However, this stimulatory effect could be demonstrated only when expression of repA and that of repB were coupled. At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an ori complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor. In contrast, the complex formed by an extract of repA-repB-expressing cells was highly specific and was resistant to both types of challenges. At a 10-fold-lower concentration, RepB did not exhibit ori binding activity, but it could nevertheless form a salt-resistant ori complex in vitro, provided that host factors were present. Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from E. coli cells expressing the repA and repB genes and also from mycobacterial cells harboring pAL5000-derived vectors. The results indicate that in vivo RepB interacts with host factors and forms an ori complex, but this activity is maximal only when there is coupled expression of repA.     

Increased expression of B-lymphocyte chemoattractant,but not pro-inflammatory cytokines,in muscle tissue in rhesus chronic Lyme borreliosis

Inflammation in skeletal muscle is a consistent feature of Lyme borreliosis, both in the human disease and experimental models. This study had two goals: to evaluate the expression of selected pro-inflammatory and chemokine genes in skeletal muscle in the Rhesus model of Lyme disease, and to identify unexpected cytokine genes involved in Lyme myositis. Two different techniques for measuring cytokine messenger RNA (mRNA) levels were used to achieve these goals: gene expression microarrays and. real-time RT-PCR (Taqman). Muscle from necropsies and biopsies were used, and were obtained from both infected and uninfected non-human primates (NHPs). Although many cytokines were found expressed in muscle tissue, pro-inflammatory cytokines commonly associated with inflammation were not consistently upregulated in infected muscles relative to uninfected muscles. However, B-lymphocyte chemoattractant (BLC), a chemokine implicated in the trafficking of B-cells into tissue, was increased in expression. This study is the first to extensively characterize cytokine gene expression in chronically inflamed tissue in Lyme borreliosis.     

In silico studies on bacterial xylanase enzyme: Structural and functional insight

Xylans are the second most abundant form of hemicelluloses and are the second most abundant polysaccharide in nature after cellulose. To degrade xylan, microbes produce mainly xylanase enzyme. Wide range of microorganisms like fungi, bacteria, yeast, marine algae etc. are capable of producing xylanase. Main source of xylanase is fungi but industrial production of bacterial xylanase is low cost, easy downstream process and high production rate. To understand primary, secondary and tertiary structure of xylanase, in silico composition of amino acids, basic physiological characteristics; viz., pI, molecular weight, instability index, GRAVY, molar extinction coefficient, secondary structure, presence of functional domain and motifs, phylogenetic tree, salt bridge compositions are determined. In silico study of xylanase focused on 36 different bacterial sources are performed by retrieving FASTA and PDB sequences using RCSB PDB. FASTA and PDB files are proceed further in ExPASy-ProtParam, RAMPAGE, QMEAN, MEME, PSIPRED, InterProScan, MOTIF scan, ERRAT, Peptide cutter, ESBRI and MEGA 7. The instability index range (16.90–38.78) clearly indicates that the protein is highly stable. α-helix mean value (27.11%) infers the protein is dominated by α-helix region. The aliphatic index (39.80–90.68) gives information that the protein is highly thermostable, prevalence by alanine amino acid in aliphatic side chain. No transmembrane domain was found in the protein which confirms the enzyme is extracellular in nature. Ancestor chart analysis confirmed that it is a part of carbohydrate metabolic process and more specifically a member of glycoside hydrolase super family.     

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100 μM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)₂]Cl₂, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)₃]Cl₃, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl₂·2H₂O) or cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) alone had no effects as “free” cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes.

Osteology,myology and feeding mechanism of Astronotus ocellatus (Pisces,Perciformes)

Summary Astronotus ocellatus captures its prey by creating a negative pressure in the buccal cavity which is caused by its quick expansion. Once the prey has been accommodated, the buccal cavity undergoes a compression which may propel the prey towards the pharyngeal jaws for mastication. The motion picture recordings indicate retracted premaxillae at the beginning of food intake followed by a maximum attainment of mouth gape and then mastication. During the maximum opening of the mouth the premaxillae are protruded and dentaries are at maximum depression. These events are followed by activities such as buccopharyngeal cavity expansion, bulging on the ventral surface of the head, and prominent curvature on the ventral surface anterior to the urohyal, caused by the upward movement of the glossohyal. Based on the cinematographic results, it may be inferred that the maximum mouth gape is caused by the sternohyoid-hyoid-interopercular-mandible coupling, and not by the opercular apparatus-mandible coupling, as the latter acts after the full descent of the lower jaw. Impression of the expanded buccopharyngeal cavity has been made by a paraffin mold technique, which confirms the displacement of the buccopharyngeal elements during expansion of the cavity.     

Isolation and characterization of non-neuronal enolase (NNE) from Neurospora crassa and comparison with neuron specific enolase isolated from neuroblastoma cell line NG108

Enolase is a vital enzyme of the glycolytic pathway. It exists mainly in two forms, non-neuronal enolase (NNE) and neuron specific enolase (NSE). Neurospora crassa, a filamentous fungus, was used as the source of pure NNE, and by using DEAE-cellulose and a Sephadex G-150 column chromatography highly purified enzyme (20.4 fold purification with 54.7 percent recovery) was obtained. The development profile of the enzyme shows a peak value after 90 hours of mycelial growth from conidia of N. crassa. In this respect, it differs from neuroblastoma NSE where the peak value of the enzyme activity appears 7 1/2 hours after the splitting of the cells. N. crassa enolase (NNE) is more thermolabile than NG108 NSE and N. crassa enolase is more sensitive to urea, chloride, and fluorophosphate. The Km values for 2-phosphoglycerate and Mg++ were 0.34 mM and 0.47 mM, respectively, for N. crassa enolase, whereas these values were 1.1 mM and 3.1 mM, respectively, in the case of neuroblastoma NSE. N. crassa enolase is a dimer molecule of molecular weight 85,000 daltons. N. crassa enolase is not neutralized by NSE antisera and neutralized by NNE antisera as opposed to neuroblastoma NSE.     

Early generation intermating for yield improvement in groundnut (Arachis hypogaea L.)

Summary Seeds of 4 crosses of groundnut (Arachis hypogaea L.), Robut 33-1 x Chico, Robut 33-1 x NC Ac 17090, Robut 33-1 x PI 298115 and MK 374 x GAUG 1, were irradiated with 30 kR. In the F₁, some branches of each plant were intermated with other plants at random and others selfed in each cross to produce S₂ and F₂ seeds. They were evaluated for pod yield, shelling percentage and 100-kernel weight. The frequency of plants superior to F[in1] was much higher in S₂ than in F₂, which was, in general, true for the values of yield and its components. The S₂ and F₂ were advanced to third generation by selfing. The families descending from S₂ showed clear superiority over those from F₂. The reason for such superiority was suggested to be the recombination of genes from the upper and lower ends of the genotypic distribution under intermating.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.