Perturbation of syntrophic isobutyrate and butyrate degradation with formate and hydrogen

The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain T1N, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobutyrate degradation by the culture. The reversible isomerization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isomerization was coupled with syntrophic butyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure (>2000 Pa) or a measurable level of formate (10 muM or higher). Significant formate (more than 1 mM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. (c) 1996 John Wiley & Sons, Inc.     

Role of erythrocytes in leukocyte-endothelial interactions: mathematical model and experimental validation.

The binding of circulating cells to the vascular wall is a central process in inflammation, metastasis, and therapeutic cell delivery. Previous in vitro studies have identified the adhesion molecules on various circulating cells and the endothelium that govern the process under static conditions. Other studies have attempted to simulate in vivo conditions by subjecting adherent cells to shear stress as they interact with the endothelial cells in vitro. These experiments are generally performed with the cells suspended in Newtonian solutions. However, in vivo conditions are more complex because of the non-Newtonian flow of blood, which is a suspension consisting of 20-40% erythrocytes by volume. The forces imparted by the erythrocytes in the flow can contribute to the process of cell adhesion. A number of experimental and theoretical studies have suggested that the rheology of blood can influence the binding of circulating leukocytes by increasing the normal and axial forces on leukocytes or the frequency of their collision with the vessel wall, but there have been no systematic investigations of these phenomena to date. The present study quantifies the contribution of red blood cells (RBCs) in cell capture and adhesion to endothelial monolayers using a combination of mathematical modeling and in vitro studies. Mathematical modeling of the flow experiments suggested a physical mechanism involving RBC-induced leukocyte dispersion and/or increased normal adhesive contact. Flow chamber studies performed with and without RBCs in the suspending medium showed increases in wall collision and binding frequencies, and a decrease in rolling velocity in the presence of erythrocytes. Increased fluid viscosity alone did not influence the binding frequency, and the differences could not be attributed to large near-wall excesses of the lymphocytes. The results indicate that RBCs aid in the transport and initial engagement of lymphocytes to the vascular wall, modifying the existing paradigm for immune cell surveillance of the vascular endothelium by adding the erythrocyte as an essential contributor to this process.     

Production of Xylanase by Thermophilic Melanocarpus albomyces IIS-68

Melanocarpus albomyces IIS-68, a thermophilic fungus was used for the production of extracellular xylanase on various agroresidues in solid-state fermentation (SSF). Growth on untreated wheat straw and sugar cane bagasse supported xylanase production, while rice straw and rice husk did not. Alkali treatment and acid chlorite treatment of these latter substrates, which lead to extensive delignification, enhanced xylanase production. In contrast, these treatments caused a decline in xylanase activity on wheat straw and bagasse. Acetyl esterase was produced concurrently with xylanase, maximal activity being produced on bagasse. Enzyme production was higher in SSF than in submerged fermentation (SmF). Studies with electron micrographs indicated that culture filtrate proteins were able to degrade wall polymers.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

Anaerobic Degradation of Normal- and Branched-Chain Fatty Acids with Four or More Carbons to Methane by a Syntrophic Methanogenic Triculture

Syntrophic degradation of normal- and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum T1N, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methylvalerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded.     

Biological, Serological and Coat Protein Properties of a Strain of Turnip Mosaic Virus Causing a Mosaic Disease of Brassica campestris and B. juncea in India

Biological, serological and coat protein properties of a potyvirus (Poty-Rape) causing a mosaic disease of Brassica campestris and B. juncea in India were investigated. The virus readily infected 4 of the 5 plant species in the family Brassicaceae in which it induced severe systemic mosaic symptoms; it also induced chlorotic and necrotic local lesions in Chenopodium amaranticolor , but failed to infect 4 other species of Chenopodiaceae or 20 species of Amaranthaceae, Apiaceae, Canabinaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Leguminosae and Solanaceae. The virus was transmitted in a non-persistant manner by Myzus persicae, Brevicoryne brassicae and Aphis gossypii. The Average size, of the virus particles in a purified preparation was 740 nm × 12 nm. SDS-PAGE analysis of the viral coat protein showed two major bands of approximately 37 kDa and 31 kDa, a pattern very similar to that of a reference isolate of turnip mosaic virus (TuMV) from the U.S. In Western-blot immunoassay, an antiserum to TuMV reacted with both the coat protein bands of the Poty-Rape islate and the reference TuMV, but not with the coat proteins of four other potyviruses. The high performance liquid chromatographic profile of tryptic peptides from the coat protein of Poty-Rape was found to be very similar to that of the reference TuMV, but differed substantially from those of four other potyviruses. The Poty-Rape isolate is considered to be a distinct strain of, TuMV.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.      

In Vitro,In Silico,ADME and Theoretical Analysis of Mn(II) and Co(II) Complexes Derived from Methyl-(Z)-N′-Carbamothioylcarbamohydrazonate Schiff Base Ligand

Mn(II) and Co(II) complexes of methyl-(Z)−N′-carbamothioylcarbamohydrazonate Schiff base ligand were synthesized. The ligand and metal salts were taken in 2 : 1 stoichiometric ratio. All the synthesized complexes were characterized using elemental analysis, molar conductance, magnetic moment and various spectroscopic techniques (FT-IR, UV/VIS, EPR) techniques. Elemental and spectroscopic results verified bidentate donor nature of the ligand and octahedral geometry of all the complexes. The non-electrolytic nature of Mn(II) and Co(II) complexes were suggested by conductivity data analysis. In vitro antibacterial (E. coli and S. aureus) and antifungal (C. albicans and C. tropicalis) screening were achieved by employing agar well diffusion method which revealed better antimicrobial activity of Co(II) complexes than Mn(II) complexes. In silico SwissADME study predicted the drug-likeness probability of ligand and complexes. The interaction of two bacterial proteins (E. coli and S. aureus) with compounds was also analyzed using molecular docking study, which corroborate the in vitro analysis.