Selective solubilization of xylan in pulp using a purified xylanase fromTrichoderma harzianum

Summary The specificity of action of a cellulase-free xylanase preparation on pulp fibers was revealed by the composition of the solubilized products after enzyme treatment. The neutral carbohydrates released by the treatment of two hardwood kraft pulps were composed exclusively of xylooligomers. A similar treatment of Solka Floc showed no detrimental effect on the degree of polymerization of the cellulose fibers, as determined by size exclusion chromatographic analysis.     

Morphological characterization of cultured bovine aortic endothelial cells and the effects of atriopeptin II and sodium nitroprusside on cellular and extracellular accumulation of cyclic GMP

Primary, first and second passaged endothelial cells from bovine aorta were grown in plastic culture dishes or on glass coverslips. The cells were characterized by their monolayer cobblestone appearance at confluence, their immunofluorescent staining for factor VIII-related antigen, their specific uptake of low density lipoprotein and by their ultrastructure. Following stimulation of the cells by atriopeptin II or sodium nitroprusside, both cellular and extracellular cyclic GMP levels were measured. Cellular cyclic GMP content was increased greatly by atriopeptin II in a time-dependent manner while sodium nitroprusside was essentially without effect. Increases in tissue cyclic GMP levels were associated with a time-dependent accumulation of the nucleotide in the extracellular compartment. Zaprinast, a specific inhibitor of cyclic GMP phosphodiesterases, did not significantly affect either basal or atriopeptin II-stimulated increases in cyclic GMP content, nor extracellular accumulation of the nucleotide. It is concluded that the cyclic GMP content of endothelial cells is not solely dependent on degradation by phosphodiesterases but also involves release of cyclic GMP into the extracellular compartment.     

Effects of temperature and moisture during incubation on carcass composition of hatchling snapping turtles (Chelydra serpentina)

Summary Flexible-shelled eggs of common snapping turtles (Chelydra serpentina) were incubated on each of two substrates (vermiculite, sand) at each of three temperatures (26.0°C, 28.5°C, 31.0°C) and three moisture regimes (wet, intermediate, dry). Embryos developing in cool, wet environments mobilized the largest amounts of protein from their yolk and attained the largest size before hatching, whereas turtles developing in warm, dry environments mobilized the smallest quantities of protein and were the smallest in body size at hatching. Embryos on wet substrates mobilized more lipid from their yolk than did embryos on dry media, but ambient temperature had no demonstrable influence on patterns of lipid mobilization. The total reserve of neutral lipid available in residual yolk plus carcass to sustain neonates in the interval prior to the beginning of feeding was largest in hatchlings from dry environments and smallest in animals from wet environments, but was unaffected by temperature during incubation. Hydration of tissues in hatchlings was higher when incubation was in cool, moist conditions than when incubation was in warm, dry settings, thereby indicating that some of the effects of moisture and temperature on mobilization of nutrients by embryos may be mediated by differences in intracellular water. Patterns of response to temperature and moisture recorded for turtles emerging from eggs on sand were similar to those recorded for hatchlings on vermiculite, so no important conclusion would have been affected by incubating eggs on one medium instead of the other.     

Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme

An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction.     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

Effects of cage beddings on microsomal oxidative enzymes in rat liver

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Specific immune regulation of chronic-relapsing experimental allergic encephalomyelitis in mice

These studies were designed to examine immunologic means of regulating the clinical course of murine chronic-relapsing experimental allergic encephalomyelitis (R-EAE). We asked whether induction of specific immune tolerance to the major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), could inhibit the development of R-EAE. Neuroantigen-specific tolerance was induced in SJL/J mice in a dose-dependent manner by the i.v. injection of mouse spinal cord homogenate-coupled syngeneic splenocytes (MSCH-SP) on day -7 relative to immunization on days 0 and +7. Sham-tolerized controls developed significant MBP- and PLP-specific DTH responses before the onset of clinical R-EAE. In contrast, MSCH-SP tolerized mice exhibited a dramatically reduced incidence of clinical and histologic signs of disease which correlated with the failure to develop MBP- and PLP-specific DTH responses. In 10 separate experiments, 118/149 (79%) of control mice, but only 22/137 (16%) of tolerized mice developed clinical R-EAE. Tolerance took time to develop and lasted at least 4 wk as mice injected with Ag-coupled splenocytes on day -1 relative to immunization remained susceptible to R-EAE, whereas mice injected on days -7, -14, or -28 were resistant. Tolerance induction required neuroantigens as injection of splenocytes coupled with a syngeneic mouse kidney homogenate failed to significantly alter the incidence of R-EAE or the development of neuroantigen-specific DTH responses. Thus, induction of R-EAE can be specifically and significantly regulated after the i.v. injection of splenocytes coupled with a crude, heterogeneous mixture of neuroantigens (i.e. MSCH).     

Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides. Correlation of effect with biologic activity of IL-2

Current malaria vaccine strategies focus on subunit vaccines that contain one or a limited number of malaria Ag. However, there is widespread nonresponsiveness to many of these Ag probably resulting from Ir gene control. Using a congenic mouse model, we demonstrated that human rIL-2 (as an adjuvant) can overcome Ir gene controlled low immune responsiveness to peptide malaria Ag vaccine candidates [R32tet32, R32LR, and Th2R-NP (NANP)5NA] as determined by the antibody response, providing it is emulsified with the Ag during immunization. This effect is not caused by IL-2 merely acting as a foreign protein and stimulating noncognate help; it requires biologic activity of the IL-2, as determined by studying the effect of inactive rIL-2, which has minimal biologic activity but which has retained its antigenicity. IL-2 does not appear to be working by an effect on priming of specific Th, and IL-2 cannot overcome an Ir gene controlled low T cell proliferative response. IL-2 may have a role to play in human vaccine development where a high titer antibody response to a subunit vaccine is required.