A New Cold-Adapted,Organic Solvent Stable Lipase from Mesophilic Staphylococcus epidermidis AT2

The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6–9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca²⁺, Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.     

Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes.

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.     

Amyloid P-component (protein AP) in localized amyloidosis as revealed by an immunocytochemical method

Summary Localization of protein AP, which is known to be associated with all amyloid-laden tissues in systemic amyloidoses, was studied by an immunocytochemical peroxidase-antiperoxidase staining method in a series of localized amyloidosis, i.e. islet amyloid, lichen amyloidosus, and nodular amyloidosis of the respiratory and urinary tracts. The amyloid fibrils of these localized amyloidoses are believed to be of three different chemical classes belonging to the AE, AD and AL type, respectively. The present study revealed that protein AP was present in amyloid deposits in all tissues examined, thus supporting that protein AP is present in amyloid deposits not only of all types of systemic amyloidosis but also different forms of localized amyloidosis.Supported by the Swedish Medical Research Council (Project No. B81-12X-05941-01), the Research Fund of King Gustaf V and grants from the United States Public Health Service, National Institute of Arthritis, Metabolism and Digestive Diseases (AM 04599 and AM 07014), National Institute of Health Multipurpose Arthritis Center (AM 20613), from the General Clinical Research Centers Branch of the Division of Research Resources, National Institutes of Health (RR 533), from the Massachusetts Chapter of the Arthritis Foundation and from the Arthritis Foundation     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of some Hedyotis species

The antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of seven Hedyotisspecies were investigated. The antioxidant activity was evaluated by the ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods while the radical scavenging activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The anti-inflammatory activity related to NO inhibition of the plant extracts was measured by the Griess assay while cytotoxicity were measured by the MTT assay against CEM-SS cell line. The antibacterial bioassay (against 4 bacteria, i.e. Bacillus subtilis B28 (mutant), Bacillus subtilis B29 (wild-type), Pseudomonas aeruginosa UI 60690 and methicillin resistant Staphylococcus aureus, (MRSA) was also carried out using the disc-diffusion method. All tested extracts exhibited very strong antioxidant properties when compared to Vitamin E (alpha-tocopherol) with percent inhibition of 89-98% in the FTC and 60-95% in the TBA assays. In the DPPH method, H. herbacea exhibited the strongest radical scavenging activity with an IC50 value of 32 microg/ml. The results from the Griess assay showed that the tested extracts are weak inhibitors of NO synthase. However, all tested extracts exhibited moderate cytotoxic properties against CEM-SS cell line giving CD50 values in the range of 21-41 microg/ml. In the antibacterial bioassay, the stems and the roots of H. capitellata showed moderate activity against the 4 tested bacteria while the leaves showed moderate activity towards B. subtilis B28, MRSA and P. aeruginosa only. The roots of H. dichotoma showed strong antibacterial activity against all 4 bacteria. All other extracts did not exhibit any antibacterial activity.     

Detection of farm fox and hybrid genotypes among wild arctic foxes in Scandinavia

In Scandinavia, farmed arctic foxes frequently escape from farms, raising concern about hybridization with the endangered wild population. This study was performed to find a genetic marker to distinguish escaped farm foxes from wild Scandinavian foxes. Microsatellite and mitochondrial control region variation were analyzed in 41 farm foxes. The results were compared with mitochondrial and microsatellite data from the wild population in Scandinavia. The farm foxes were genetically distinct from the wild foxes (F _ST=0.254, P < 0.00001) and all farm foxes had a single control region haplotype different from those observed in the wild population. We developed a method based on Restriction Fragment Length Polymorphism (RFLP) on the mitochondrial control region to differentiate between farmed and wild arctic foxes. This test was subsequently successfully used on 25 samples from free-ranging foxes, of which four had a suspected farm origin. All four of the suspected foxes, and none of the others, carried the farm fox haplotype. Three of these were successfully genotyped for all eleven microsatellite loci. A population assignment test and a Bayesian Markov Chain Monte Carlo analysis indicated that two of these individuals were escaped farm foxes, and that the third possibly was a hybrid between a farmed and a wild arctic fox.     

Semi-industrial Scale Production of a New Yeast with Probiotic Traits, <Emphasis Type="Italic">Cryptococcus</Emphasis> sp. YMHS,Isolated from the Red Sea

A new yeast strain with promising probiotic traits was isolated from the Red Sea water samples. The isolate (YMHS) was subjected to genetic characterization and identified as Cryptococcus sp. Nucleotide sequence analysis of the rRNA gene internal transcribed spacer regions showed 95% sequence similarity between the isolate and Cryptococcus albidus. Cryptococcus sp. YMHS exhibited desirable characteristics of probiotic microorganisms; it has tolerance to low pH in simulated gastric juice, resistance to bile salts, hydrophobic characteristics, broad antimicrobial activity, and in vitro ability to degrade cholesterol. The isolate grew well in a semi-defined medium composed of yeast extract, glucose, KH₂PO₄, (NH₄)₂SO₄, and MgSO₄, yielding cell mass of 2.32 and 5.82 g/l in shake flask and in bioreactor cultures, respectively. Fed-batch cultivation, with controlled pH, increased the biomass gradually in culture, reaching 28.5 g/l after 32 h cultivation. Beside the feasible use as a probiotic, the new strain also could be beneficial in the development of functional foods or novel food preservatives. To our knowledge, this is the first report of yeast with probiotic properties isolated from the Red Sea.