Bedding disposal cabinet for containment of aerosols generated by animal cage cleaning procedures.

Laboratory tests with aerosolized spores and animal room tests with uranine dye indicate the effectiveness of a prototype bedding disposal cabinet in reducing airborne contamination generated by cage cleaning procedures.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

Characterization of exopolysaccharides produced by rhizobacteria

Summary Bacteria isolated from the rhizosphere, the rhizobacteria, of sorghum, pearl millet, wheat, alfalfa and rice were screened for the production of exopolysaccharide (EPS). Nearly a quarter of the strains produced exopolysaccharides, either capsular or hydrosoluble slime. A majority of the isolates produced slime. Physico-chemical analyses have indicated the ability of certain diazotrophic Pseudomonas paucimobilis isolates from millets and sorghum to produce unique types of EPS, which are highly viscous and thermostable.Correspondence to: T. Heulin     

Characterization of plasminogen activator produced by an established cell line from human ovary

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.     

Characterization of a factor produced by human T cell clones exhibiting eosinophil-activating and burst-promoting activities

It has long been suggested that eosinophil response observed in certain immunological reactions depends on the release of soluble products from sensitized lymphocytes when exposed to the challenging antigen. We were able to show that alloreactive T cell clones (ATLC) obtained from human rejected kidney produced, when stimulated with specific antigen (kidney donor-B lymphoblastoid cell line) and interleukin 2, a factor triggering the proliferation of a subline (DA-2) of the interleukin 3 sensitive DA-1 murine cell line. The biochemical features of this factor called HILDA (human interleukin DA) and the DA-2 nonresponsiveness to several human T cell lymphokines and cytokines lead us to the conclusion that this 41,000 m.w. glycoprotein could not be likened to already known T cell lymphokines. Highly purified HILDA turned out to be a potent chemoattractant and activator of, respectively, mouse and human eosinophils. It also displayed burst-promoting activity on human marrow.     

Generation of large droplet aerosols within microbiological containment using a novel flow-focussing technique

Flow-focussing technology was harnessed to enable generation of large droplet aerosols within high-level microbiological containment. The Collison nebuliser and flow-focussing aerosol generator (FFAG) produced aerosols from distilled water with average mass median aerodynamic diameters (MMAD) of 4.19 and 11.93 μm, respectively. The medium type [water, phosphate buffered saline (PBS) or microbiological broth] had minimal effect on the droplet size generated by the FFAG. The FFAG can be modulated to generate reproducible aerosols with a wide range of MMADs (9–105 μm). The number of particulates (i.e. fluospheres) contained within the droplets increased as the MMAD increased from 12 to 50 μm. The technology described can be used for the exposure of small-animal models to large droplet aerosols (>10 μm) and has applications in healthcare, pharmaceutical, agricultural and biodefence environments.     

Characterization of HCV-like particles produced in a human hepatoma cell line by a recombinant baculovirus

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.     

Characterization of two bacteriocins produced by Clostridium perfringens

Two types of bacteriocins were shown to be produced in succession by a strain of Clostridium perfringens SN-17. They were separated by diethylaminoethyl cellulose (DEAE) column chromatography at pH 8.5 with a linear concentration gradient of NaCl. One type of bacteriocin (named SN-a) was eluted at 0.07 M and the other type (named SN-b) was at 0.12 M. Each of these was partially purified in a series of column chromatographies: DEAE, Sephadex G-200 (or Bio Gel P-150), and hydroxyapatite. Specific activities of SN-a and SN-b after the last chromatography were at most 30- to 50-fold that of culture filtrate of the organisms. Chromatographed SN-a migrated as a single zone in polyacrylamide gel electrophoresis (PAGE) and the zone showed high biological activity. On the other hand, PAGE pattern of SN-b revealed the presence of a few contamination materials. The activity of SN-b after the last chromatography was hardly recovered from the gel but inactivated SN-b was identified in the gel by examining bacteriocin activity of the DEAE fractions recovered from the gel. The molecular weight of the SN-a and SN-b was determined to be about 70,000 and 100,000, respectively, by molecular sieve chromatography. These bacteriocins were very sensitive to protease but insensitive to DNase and RNase. Bacteriocins were both completely inactivated at 55 C and they were more stable in alkaline pH than in acidic pH. SN-a and SN-b were adsorbed in different ways on the surface of the producer and insensitive strains. Several differences and similarities between these 2 bacteriocins are discussed with special reference to the relationship between them.     

Characterization of bacteriocin 28 produced by Clostridium perfringens

Bacteriocin 28, produced by Clostridium perfringens, was characterized by gel filtration and sodium dodecyl sulfate - polyacrylamide gel electrophoresis as a glycoprotein with a molecule weight of approximately 100,000. Density gradient centrifugation suggested a lower weight of 84,000. The bacteriocin bound firmly to phenyl-Sepharose CL-4B gel, indicating hydrophobic properties, and elution from this gel with ethylene glycol clearly separated bacteriocin from the alpha and theta toxins of C. perfringens, the latter of which was also hydrophobic. Bacteriocin 28 was immunogenic, inducing neutralizing and precipitating antibodies, and possessed three isoelectric points: 7.37, 7.05, and 5.4. Amino acid and carbohydrate analysis of the active material showed a composition of 15 amino acids and several carbohydrates. The molecule demonstrated instability with increasing purification, and several approaches to purification are described.     

Characterization of a pilus produced by Aeromonas hydrophila

A pilus produced by Aeromonas hydrophila was purified and partially characterized. The pilin monomers had an apparent molecular weight of 17,000. Agglutination studies indicated serological cross-reactivity in the pili of A. hydrophila strains. Presence of pili did not correlate with hydrophobicity or haemagglutinating ability of the bacteria.     

Characterization of levan produced by Serratia sp.

Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus.     

Characterization of laccase activity produced by Cryptococcus albidus

This study deals with the characterization of laccase enzyme activity produced by Cryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a number of chemicals. These chemicals can interfere with the proper functioning of the enzymes used for bioremediation. Thus, it is important to study the effect of such interfering solvents, detergents, metal chelators, and other chemicals on enzyme activity before industrial applications. Laccase showed maximum activity at pH 2.5 and temperature 20-30°C when ABTS was used as a substrate. The enzyme followed Michaelis-Menten kinetics: K(m) was 0.8158 mM and V(max) was 1527.74 U/mg. Laccase showed good thermostability with a half-life of 81 min at 25°C, 77 min at 35°C, 64 min at 45°C, 36 min at 55°C, and 21 min at 65°C. There was no effect of sodium dodceyl sulfate (SDS) (0.1-1.0%) and EDTA (0.1-0.5%) on laccase activity. Sodium azide and 2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lower concentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, the activity was completely inhibited at a concentration of 50% or above of acetone, methanol, 1,4-dioxan, and acetonitrile.     

Characterization and partial purification of a non-interferon macrophage activating factor produced by human leukemic T cell line

Culture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-gamma, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-gamma, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-gamma monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40,000 to 60,000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1,300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-gamma.     

Characterization and biobleaching effect of hemicellulases produced by thermophilic fungi

The culture supernatant of Thermomyces lanuginosus strains MED 2D and MED 4B1 had high activities of xylanase with low inducible activities of -xylosidase. The crude xylanase was optimally active at 70 °C and at pH 6.0 to 6.5. Subsequently Eucalyptus kraft pulp was treated with MED 2D supernatant at 10 IU per gram pulp resulting in a 10.5% reduction in Kappa number. XECEDED-Refined bleached pulp resulted in handsheets with increased brightness compared to the control (X = xylanase treatment; E = alkaline extraction; C = Cl2 treatment; D = ClO2 treatment).     

Characterization of rhamnolipids produced by wild-type and engineered Burkholderia kururiensis

Biosurfactants are a class of functional molecules produced and secreted by microorganisms, which play important roles in cell physiology such as flagellum-dependent or -independent bacterial spreading, cell signaling, and biofilm formation. They are amphipathic compounds and comprise a variety of chemical structures, including rhamnolipids, typically produced by Pseudomonas spp. and also reported within other bacterial genera. The present study is focused on Burkholderia kururiensis KP23^T, a trichloroethylene (TCE)-degrading, N-fixing, and plant growth-promoting bacterium. Herein, we describe the production of rhamnolipids by B. kururiensis, and its characterization by LTQ-Orbitrap Hybrid Mass Spectrometry, a powerful tool that allowed efficient identification of molecular subpopulations, due to its high selectivity, mass accuracy, and resolving power. The population of rhamnolipids produced by B. kururiensis revealed molecular species commonly observed in Pseudomonas spp. and/or Burkholderia spp. In addition, this strain was used as a platform for expression of two Pseudomonas aeruginosa biosynthetic enzymes: RhlA, which directly utilizes β-hydroxydecanoyl-ACP intermediates in fatty acid synthesis to generate the HAA, and RhlB, the rhamnosyltransferase 1, which catalyzes the transfer of dTDP-L-rhamnose to β-hydroxy fatty acids in the biosynthesis of rhamnolipids. We show that rhamnolipid production by the engineered B. kururiensis was increased over 600 % when compared to the wild type. Structural analyses demonstrated a molecular population composed mainly of monorhamnolipids, as opposed to wild-type B. kururiensis and P. aeruginosa in which dirhamnolipids are predominant. We conclude that B. kururiensis is a promising biosurfactant-producing organism, with great potential for environmental and biotechnological applications due to its non-pathogenic characteristics and efficiency as a platform for metabolic engineering and production of tailor-made biosurfactants.