SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3β (GSK-3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3β at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3β, and an increase in the phosphorylation of tau. K_m of calcineurin for a GSK-3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3β were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3β. We conclude that in vitro, calcineurin can dephosphorylate GSK-3β at Ser-9 and form a stable complex with GSK-3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3β in vivo .     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.     

Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline-Directed Protein Kinases or Cyclic AMP-Dependent Protein Kinase

Abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein τ. Previous studies have identified serine and threonine residues phosphorylated in PHF-τ and have shown that τ can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant τ phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3β, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates τ phosphorylated in this manner.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Effect of heat shock on DNA-dependent RNA polymerase from the cyanobacteriumSynechococcus sp.

Purification of DNA-dependent RNA polymerase from exponentially growing cells of the cyanobacteriumSynechococcus sp. is described in cultures grown at normal temperature (39°C) and after heat shock (HS) (47°C). Polyethyleneimine precipitation followed by chromatography and gel filtration steps results in a 39% yield. The enzyme has a component of molar mass of 43 kDa, designated σ, in addition to the typical procaryotic β’β∝₂ and γ. The results suggest thatSynechococcus RNA polymerase is similar to that of cyanobacterial andE. coli RNA polymerases. Electrophoresis of the HS preparation showed that the enzyme has a component of 18 kDa. This suggests the existence of a functional relationship between this protein and the HS response ofSynechococcus RNA polymerase, probably in salvaging denatured RNA polymerase or helping to regain its native structure.     

Further studies on eukaryote DNA stimulation of amino acid incorporation in E. coli extracts

DNA from adenovirus-2 and mouse myeloma tumors stimulate RNA synthesis and amino acid incorporation into protein in a cell-free extract from Escherichia coli. The RNA synthesis is dependent on exogenous DNA, and the RNA can be hybridized to respective template DNA. A major part of this RNA is also found attached to E. coli polysomes suggesting that RNA with messenger-like activity has been synthesized. However, the in vitro-synthesized polypeptides using adenovirus DNA or myeloma DNA do not correspond in size or antigenic activity to either the virion proteins or immunoglobulins, respectively.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Rab2 requires PKC iota/lambda to recruit beta-COP for vesicle formation

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre-Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC-like protein to recruit β-COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre-Golgi intermediates. J Biol Chem 1998;273: 17269–17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose-dependent manner. Microsomes treated with anti-PKCι/Λ lost the ability to bind β-COP, suggesting that Rab2 requires PKCι/Λ for β-COP recruitment. The recruitment of β-COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2-mediated vesicle budding. We found that the addition of either a kinase-deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v ¯esicular t ¯ubular c ¯lusters (VTCs), which promotes the recruitment of COPI to generate retrograde-transport vesicles.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

Cloning of an Outer Membrane Protein Gene from Campylobacter jejuni

An antigen in the outer membrane protein (OMP) fraction of Campylobacter jejuni was identified and characterized. Western blot analysis demonstrated antigenic differences in this protein between two congenic C. jejuni strains. Strain A74/C, which colonizes chickens, expressed the antigen at 34 kDa, while strain A74/O, which poorly colonizes chickens, expressed the antigen at 32 and 34 kDa. A genomic library was constructed in λgt11 with DNA from A74/O and screened with antibody raised against C. jejuni OMPs. A clone that possessed a 1.3-kb insert and expressed an immunoreactive protein fused to β-galactosidase was isolated and purified. DNA sequence analysis revealed the insert contained one open reading frame 864 bases long. The deduced amino acid sequence demonstrated 56.3% similarity with Bacillus steorothermophilus glnH, a glutamine-binding protein, and 54.0% similarity with C. jejuni PEB1, a putative colonization adhesin. Southern hybridization, Northern hybridization, and DNA sequence analyses of the congenic colonizing and noncolonizing strains of C. jejuni failed to distinguish the two strains and revealed only one copy of the gene. Post-translational modification may be an alternate explanation for the antigenic differences seen between the two strains. Received: 15 October 1996 / Accepted: 3 December 1996     

Partial characterization of a natural agglutinin in the hemolymph of the lobster, Homarus americanus

Partial characterization of a natural agglutinin in the hemolymph of the lobster showed: (1) Calcium ion stabilized the agglutinin against inactivation at moderate (35–45°C) temperatures as well as against a reversible increase in activity at nonphysiological pH levels (pH 6 and 9). (2) Serum frozen at ?20°C in the absence of calcium showed an increase in agglutinin activity. (3) Heat inactivation curves indicated a single component for the agglutinin. (4) Heat inactivation destroyed the ability of the agglutinin to be adsorbed to the erythrocyte antigen. (5) The chemical nature of the antigenic reactive site differed with the species of erythrocyte tested; D-glucosamine blocked or inhibited adsorption of the agglutinin to erythrocytes of most species tested. (6) In vivo the agglutinin is located in the hemocytes.     

Endosomal Sorting of Amyloid Precursor Protein-P-Selectin Chimeras Influences Secretase Processing

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α-secretase resides, or moves to acidic compartment(s) for β-secretase exposure. While the trans-Golgi network contains β-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less α-secretase cleavage, and more β-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to α- and β-secretase activities, and suggest that, because delivery to late endocytic compartments favors β-secretase processing of amyloid precursor protein, there is likely limited β-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.     

GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3β (GSK3β) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3β. Two different inhibitors of GSK3β (lithium and GSK3β-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3β is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.     

Effect of protein phosphorylation on the activity of RNA polymerase in streptomycetes

RNA polymerase was isolated fromStreptomyces granaticolor and protein kinase was partially purified fromStreptomyces albus. When RNA polymerase was treated with protein kinasein vitro the activity of RNA polymerase was markedly enhanced. Furthermore, a protein ofM=65 kDa was isolated which, after being phosphorylated, stimulated RNA polymerase activityin vitro. Because neither the β-subunits nor the α-subunits of RNA polymerase were phosphorylated it is assumed that phosphorylation of the 65 kDa protein may regulate the activity of RNA polymerase in streptomycetes.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.     

A novel metagenome-derived β-galactosidase: gene cloning, overexpression, purification and characterization

A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na⁺, K⁺, or Ca²⁺ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.