Synthesis,characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from <Emphasis Type="Italic">Streptomyces xinghaiensis</Emphasis> OF1 strain

We report synthesis of silver nanoparticles (AgNPs) from Streptomyces xinghaiensis OF1 strain, which were characterised by UV–Vis and Fourier transform infrared spectroscopy, Zeta sizer, Nano tracking analyser, and Transmission electron microscopy. The antimicrobial activity of AgNPs alone, and in combination with antibiotics was evaluated against bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, and yeasts viz., Candida albicans and Malassezia furfur by using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum biocidal concentration of AgNPs against bacterial and yeast strains were determined. Synergistic effect of AgNPs in combination with antibacterial and antifungal antibiotics was determined by FIC index. In addition, MTT assay was performed to study cytotoxicity of AgNPs alone and in combination with antibiotics against mouse fibroblasts and HeLa cell line. Biogenic AgNPs were stable, spherical, small, polydispersed and capped with organic compounds. The variable antimicrobial activity of AgNPs was observed against tested bacteria and yeasts. The lowest MIC (16 µg ml^?1) of AgNPs was found against P. aeruginosa, followed by C. albicans and M. furfur (both 32 µg ml^?1), B. subtilis and E. coli (both 64 µg ml^?1), and then S. aureus and Klebsiella pneumoniae (256 µg ml^?1). The high synergistic effect of antibiotics in combination with AgNPs against tested strains was found. The in vitro cytotoxicity of AgNPs against mouse fibroblasts and cancer HeLa cell lines revealed a dose dependent potential. The IC₅₀ value of AgNPs was found in concentrations of 4 and 3.8 µg ml^?1, respectively. Combination of AgNPs and antibiotics significantly decreased concentrations of both antimicrobials used and retained their high antibacterial and antifungal activity. The synthesis of AgNPs using S. xinghaiensis OF1 strain is an eco-friendly, cheap and nontoxic method. The antimicrobial activity of AgNPs could result from their small size. Remarkable synergistic effect of antibiotics and AgNPs offer their valuable potential in nanomedicine for clinical application as a combined therapy in the future.     

Identification of a Novel Marker for Primordial Smooth Muscle and Its Differential Expression Pattern in Contractile vs Noncontractile Cells

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375–392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, M_r = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, M_r = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle α-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle α-actinin. Our results suggest that the 1E12 antigen is a member of the α-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.     

Scanning and transmission electron microscopic observations of the cloacal epithelia of the domestic fowl

Summary The epithela of the three divisions (coprodaeum, urodaeum, proctodaeum) of the cloaca of the hen, and of the excretory ducts (colon, ureter, vagina) which join the divisions, are described using light microscopy, and scanning and transmission electron microscopy. Each region of the cloaca has its typical epithelium. Special attention is focussed in this study on the boundaries between the different epithelia. The coprodaeal epithelium does not differ considerably from that of the colon; a transitional zone is not visible. Distinct border zones, however, are observed between the other regions (ureter — urodaeum; vagina — urodaeum and proctodaeum; urodaeum-proctodaeum; proctodaeum — cutis). Although the vaginal opening is generally thought to lie in the urodaeum, our investigations show that at the vaginal opening into the cloaca the ciliated epithelium changes, on one border to a secretory epithelium characteristic of the urodaeum and on the other border to that characteristic of the proctodaeum. These observations are discussed in relation to functional aspects.     

Stimulation of juvenile hormone biosynthesis by analogues of a Manduca sexta allatotropin: in vitro studies.

Two analogues of a Manduca sexta allatotropin (Mas-AT) were synthesized. They correspond to the active fragment, amino acids 5-13, of the natural Mas-AT with substitution of norleucine for methionine. ATANA has the structure Val-Glu-Nle-Nle-Thr-Ala-Arg-Gly-Phe-NH2, ATAA is acetylated at the N-terminus. Allatotropic potency was evaluated by measuring the in vitro rates of juvenile hormone (JH) biosynthesis in corpora allata (CA) of M. Sexta. At a concentration of 20 nM, ATANA and ATAA increased JH production in day 0, day 1, and day 3 adult female CA by a factor of 3-8. Larval CA were not affected. These results correspond to activities reported for the natural Mas-At. ATANA did not stimulate pharate adult female CA to produce JH. Stimulation of female CA with ATANA was reversed when the CA were transferred to fresh medium while stimulation with ATAA under the same condition persisted. Exogenous farnesoate was converted to JH-III at a rate exceeding the highest ATANA-stimulated rate. ATANA in addition to farnesoate did not increase JH-III production, but increased JH-II production in addition to the already high production rate for JH-III. It is inferred that Mas-AT stimulates a rate-limiting enzyme in the biosynthesis of farnesoate and its homologues but does not affect epoxidation and methylation.     

The regulation of intramuscular nerve branching during normal development and following activity blockade

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.     

Synthesis from pullulan of spacer-arm, lipid, and ethyl glycosides of a tetrasaccharide [alpha-D-Glc-(1----6)-alpha-D-Glc-(1----4)-alpha-D-Glc-(1----4)-D-Glc] found in human urine; preparation of neoglycoproteins

Enzymic hydrolysis of pullulan, followed by acetylation and chromatography, gave acetylated alpha-D-Glcp-(1----6)-alpha-D-Glcp-(1----4)-alpha-D-Glcp-(1----4)-D-Glcp which, with 2-bromoethanol and boron trifluoride etherate in dichloromethane, gave the 2-bromoethyl glycoside. The reactions of the glycoside with methyl 3- mercaptopropionate , methyl 11- mercaptoundecanoate , and octadecanethiol are described, and also its hydrogenolysis to give an ethyl glycoside. The mercaptopropionate -derived, spacer-arm glycoside has been coupled to bovine serum albumin and keyhole limpet haemocyanin.     

Generic composition and physiological and cultural properties of heterotrophic bacteria isolated from soil, rhizosphere and mycorrhizosphere of pine (Pinus silvestris L.)

Among the bacteria studied Arthrobacter globiformis was predominating in the root zone, while in the non-rhizosphere soil most numerous were Bacillus circulans and A. globiformis. Ammonifiers were more numerous among the root zone bacteria than among the root free soil organisms. The reverse was noted with bacteria capable of hydrolysing starch, cellulose, pectin and chitin.     

Metagenomic analysis of a stable trichloroethene-degrading microbial community

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.