Enzyme immobilization on fibrin.

The following conclusions can be drawn concerning the utilization of fibrin to immobilized enzyme systems. Fibrin can be used both as a powder or membrane, to covalently immobilize trypsin with retention of activity. Carbon-14 labeled trypsin can be used to estimate the amount of immobilized enzyme on a proteinaceous support. Significant amounts of noncovalently coupled (adsorbed) enzyme are present on the surface of the support. Esterase activity of the immobilized labeled trypsin was inversely proportional to the amount of attached enzyme. Optimum TAME hydrolysis occurred at pH 8-8.4. The storage stability of trypsin was enhanced. Inhibition of trypsin esterase activity occurred at substrate concentrations greater than 30mM.     

Immune serum-mediated cytotoxicity against Trypanosoma rhodesiense.

The metabolic integrity of Trypanosoma rhodesiense can be assessed in vitro by the extent of incorporation of radiolabeled leucine into trichloroacetic acid precipitable material or into material retained after filtration on a glass fiber filter. Incorporation is an approximately linear function of time, and the rate of incorporation is linearly dependent on cell concentration in the presence of normal rat serum. Incorporation is completely prevented if the organisms are reacted wiith fresh serum from animals immunized with gamma-irradiated parasites; the degree of inhibition is a function of the dose of immune serum used. This serum-mediated cytotoxic activity is abrogated by heating the serum, but can be fully restored by addition of fresh rat or guinea pig serum to the heated immune serum. The serum activity arises promptly after one to four immunizing doses of irradiated parasites, falls to lower levels by 1 month, but persists for at least 2(1/2) months, and is unaffected by challenge with viable trypanosomes.     

Identification of a novel inositol bisphosphate isomer formed in chemoattractant stimulated human polymorphonuclear leukocytes

Analysis of inositol phosphate formation in chemoattractant-stimulated human polymorphonuclear leukocytes demonstrated the production of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4-bisphosphate and another inositol bisphosphate isomer not detected in unstimulated cells. Studies in cell sonicates provided evidence that the previously unidentified inositol bisphosphate isomer is produced via the degradation of inositol 1,3,4-trisphosphate. This unidentified inositol bisphosphate peak was purified by high pressure liquid chromatography, and base hydrolyzed to form a mixture of inositol monophosphate isomers. Based on these studies, the unidentified peak was identified as inositol 3,4-bisphosphate. Identification of this isomer defines a new metabolic product derived from the initial inositol 1,4,5-trisphosphate formation, and also suggests another substrate for the inositol 1-phosphatase.     

Molecular epidemiology and novel combinations of auxotype, serovar, and plasmid content in tetracycline-resistant Neisseria gonorrhoeae isolated in Canada

Between October 1987 and June 1989, 84 isolates of Neisseria gonorrhoeae carrying the TetM resistance determinant (TRNG) were received at the Laboratory Centre for Disease Control, Ottawa, from six Canadian provinces and were characterized into classes based on auxotype, serovar and plasmid content. One-fifth (17/84) of the TRNG were also penicillinase producing (PPNG). The PPNG-TRNG isolates comprised six classes based on auxotype, serovar, and plasmid content. Most (16/17) PPNG-TRNG carried 3.2-MDa beta-lactamase plasmids and the 25.2-MDa TetM-containing plasmid. We report, for the first time, the association of a 4.5-MDa beta-lactamase plasmid with the 25.2-MDa plasmid in a clinical TRNG isolate. Non-PPNG TRNG isolates comprised 11 classes based on auxotype, serovar, and plasmid content, including two previously unreported auxotype-serovar classes, P/IB-26 and P/IB-20.     

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.     

Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor

Polyclonal antibodies were produced for the development of competitive ELISA's and surface plasmon resonance (SPR)-based BIAcore inhibition assays for the detection of morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine). A conjugate consisting of M3G and ovalbumin was produced and used for the generation of antibodies, for the coating of immunoplates and for immobilisation onto BIAcore chips. Competition ELISA's were developed in PBS and urine to characterise the antibodies ability to recognise free M3G. SPR-based inhibition immunoassays on BIAcore were developed. The regeneration of the surface of a chip immobilised with conjugate following antibody binding, essential for the development of inhibition assays was investigated. Regeneration of the conjugate-coated surface was optimised for both polyclonal antibodies resulting in binding-regeneration capacities of approximately 60 cycles for one antibody and 50 cycles for the second antibody. The inhibition assays developed in urine had ranges of detection of 762-24,400 (antibody 1) and 976-62,500 pg ml(-1) (antibody 2). The inter-day coefficients of variation for the assays ranged from 1.48 to 11.24%.     

Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.