Glycan modification of a thermostable recombinant (1-3, 1-4)-beta-glucanase secreted from Saccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3, 1-4)- beta-glucanase H(A16-M) has been achieved in Saccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1P) and the Bacillus macerans (1-3, 1-4)-beta-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3'-phosphoglycerate kinase promoter, each in two different Saccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y.     

Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p

Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor. This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation. Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target. Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner. In addition, Pds1p is directly ubiquitinated by the Xenopus APC. In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase. We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase.     

Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.     

Structural analysis of oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Bufo bufo: characterization of the carbohydrate sequence Gal(alpha1-3)GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal

We report the isolation and extensive analysis of highly polymorphic MHC class I genes from sharks (Triakis scyllia), which belong to the most primitive vertebrate group with jaws, the cartilaginous fish. Predicted complete peptide-binding domains showed retention of the critical amino acid residues that would interact with antigenic peptide termini and revealed extensive allelic polymorphisms comparable to those of classic human MHC class I molecules. Mosaic structures were apparent in these domains, suggesting recombinational mechanisms to create allelic diversity. The present study demonstrates the establishment of the basic strategy for antigen-presentation employed by MHC class I molecules and documents complete divergence of two polymorphic MHC classes at a phylogenetically primitive stage of vertebrate evolution.     

Study of the polymorphism of ovine alpha s1- and alpha s2-caseins by capillary electrophoresis

Polymorphism of ovine alpha s-caseins was studied by capillary electrophoresis at pH 3.0 +/- 0.1. Individual caseins (CN) were selected according to their genetic variants, as determined by PAGE and isoelectric focusing. The ovine caseins, containing different genetic variants of alpha s1-CN and alpha s2-CN, were fractionated by cation-exchange FPLC. The alpha s1-CN variants A, B and C, and the fast moving alpha s2-CN variant were identified by a capillary electrophoresis method. The fast moving alpha s2-CN variant, so-called for its behaviour in PAGE, also had a faster electrophoretic mobility than the common alpha s2-CN when analysed by the present technique. The capillary electrophoresis method gave excellent, rapid, automated separation of alpha s1-CN and alpha s2-CN variants and was suitable for screening studies.     

Characterization of the Saccharomyces cerevisiae cdc42-1ts allele and new temperature-conditional-lethal cdc42 alleles

The administration of neurotrophins affects neuronal survival and growth, but less is known about their ability to modify the expression of growth associated genes following injury to CNS neurons. Here we characterize the effect of brain-derived neurotrophic factor (BDNF) on mRNA levels for T alpha1 alpha-tubulin, and for GAP-43, two genes whose expression levels in retinal ganglion cells (RGC) tend to correlate with growth. We first determined that most adult rat RGCs can retrogradely transport BDNF by injecting 125I-BDNF into RGC target sites in vivo. We then used quantitative in situ hybridization to characterize the effect of axotomy, or axotomy and BDNF administration on mRNA levels for GAP-43 and T alpha1. Axotomy alone resulted in a general decrease in T alpha1 alpha-tubulin mRNA levels by 2 weeks, and elicited an increase in GAP-43 mRNA levels in an average of 30% of surviving RGCs. The intravitreal administration of a single dose of BDNF (5 microg) to axotomized RGCs on the day of injury did not affect T alpha1 alpha-tubulin mRNA levels, but was followed by a moderate (approximately 80%), and short-lasting enhancement of GAP-43 mRNA levels in most RGCs during the first week after axotomy. No significant increase in GAP-43 mRNA levels was observed when BDNF was injected into the uninjured eye. We conclude that BDNF specifically enhances GAP-43 but not T alpha1 mRNA levels in injured RGCs. Because BDNF is known to stimulate branch length of injured RGCs, we suggest that changes in the expression of GAP-43, but not T alpha1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth.     

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae

In determining levels of expression of HIV-1 Nef protein within the central nervous system (CNS) we assessed antibody responses to the protein both peripherally and in CNS. Antibodies to Nef were not detected within the CNS despite detection of antibodies to both gp41 and Nef in peripheral blood and representative virus isolates derived from CNS and peripheral blood (PB) samples containing full length nef sequence and virus-infected cells expressing Nef protein. We conclude from this that expression of Nef within the CNS is such that little or no antibody production occurs and that these differences indicate that Nef protein may not be directly contributing to the AIDS dementia complex. Expression of Nef protein in PHA-activated peripheral blood mononuclear cells from CNS derived isolates was different to that of coincidental PB derived isolates in that partial surface expression was observed for the latter. The results suggest that antigenic presentation of Nef within the CNS is anomalous and that Nef protein expression, at least for the limited number of in vitro derived isolates tested, has a different localization pattern.     

Vacuole segregation in the Saccharomyces cerevisiae vac2-1 mutant: structural and biochemical quantification of the segregation defect and formation of new vacuoles

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741-1748] shifted to non-permissive temperature (37 degrees C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37 degrees C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37 degrees C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding.     

Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites

Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Delta apn2Delta strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase zeta mediates the mutagenic bypass of AP sites.     

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

Female wild Japanese monkeys (Macaca fuscata), as with all male cercopithecoids, use the mesiobuccal surfaces or the elongated crests of the mandibular third premolars (P3s), as cutting blocks that wear against edges of maxillary canines during threat manifestation or food-eating. In other words, the crests of their P3s are honed with the maxillary canines. The crests become sloped during growth and more heavily striated with the advance of age. The number, directions, lengths, and widths of these striations have been analyzed quantitatively using scanning electron microscopy (SEM). Two samples showed two distinct types of parallel striations, one longer and thicker (171.5 microns long and 14.5 microns wide on average) than the other (114.8 microns long and 12.0 microns wide on average). These striations were caused by contact between the sharp edge of the upper canine and the P3 during honing (canine/premolar complex). The long and thick striations are asymmetrical with widened parts or pits on one end, and were easily distinguished from other thinner striations which may have been caused by fine particles. The third sample showed Hunter-Schreger bands with striae of Retzius on the sloping heavily worn mesiobuccal surface. The features of these thick parallel striations indicate that they result from closing movements of the jaw.     

Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor

DNA released from neutrophils at sites of inflammation may modulate tissue proteolysis. We used tRNA and synthetic polynucleotides as models of DNA to study the influence of polynucleotides on the inhibition of neutrophil elastase by its endogenous inhibitors alpha1-proteinase inhibitor (alpha1-PI) and mucus proteinase inhibitor (MPI). Affinity chromatography showed that polynucleotides form electrostatic complexes with elastase and MPI but not with alpha1-PI, the highest affinity being for MPI. The tight-binding partial inhibition of elastase by polynucleotides was used to calculate the Kd of the elastase-polynucleotide complexes which ranged from 4 microM to 21 nM. One mole of tRNA was able to bind 9 mol of elastase. Polydeoxycytosine and tRNA significantly impaired the reversible inhibition of elastase by MPI: they moderately increased the rate of enzyme-inhibitor association, strongly enhanced the rate of complex dissociation, and lowered the enzyme-inhibitor affinity by factors of 34 and 134, respectively. The two polynucleotides also decreased the rate of the irreversible inhibition of elastase by alpha1-PI by factors of 30 and 3, respectively. Polynucleotides also changed the mechanism of inhibition of elastase by the two inhibitors from a one-step inhibition reaction to a two-step binding mechanism. Our data may help explain why proteolysis may occur at sites of inflammation despite the presence of active proteinase inhibitors.     

Stimulation of prostaglandin E2 production by interleukin-1 alpha and transforming growth factor alpha in osteoblastic MC3T3-E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor alpha (TGF-alpha) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [3H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1 alpha into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-fold by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha caused a 1.7-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.     

Identification of the MNN2 and MNN5 mannosyltransferases required for forming and extending the mannose branches of the outer chain mannans of Saccharomyces cerevisiae

The mannan structure found on the N-linked glycans of the yeast Saccharomyces cerevisiae is composed of a long backbone of alpha-1, 6-linked mannose to which are attached branches consisting of two alpha-1,2-linked mannoses followed by an alpha-1,3-linked mannose. In the mutants mnn2 and mnn5, the addition of the first and second of these two mannoses, respectively, is defective. In this paper, we report the identification of the genes corresponding to these mutations. The two genes encode closely related proteins with distant homology to the known Mnn1p alpha-1,3-mannosyltransferase. We show that these proteins are localized in an early compartment of the yeast Golgi and that they are not physically associated with each other or with the two protein complexes known to be involved in synthesizing the alpha-1,6-linked backbone. The identification of Mnn2p and Mnn5p allows us to assign Golgi proteins to all of the catalytic steps in S. cerevisiae mannan synthesis.