Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )     

Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase). The phosphorylation of calpastatin was investigated in human hematopoietic system cell lines. Microheterogeneity of calpastatin was observed, in which 118- and 116-kDa forms were named calpastatin a and b, respectively. The phosphorylation of both calpastatins was identified in all cell lines examined and occurred mainly at serine residues with trace amounts of phosphothreonine in vivo. The incubation of cells with 12-O-tetradecanoylphorbol-13-acetate increased the incorporation of 32P-orthophosphate into calpastatin a. Two-dimensional maps of 32P-labeled phosphopeptide from both calpastatins were identical except for additional minor spots for calpastatin a. [35S]methionine-labeled calpastatins a and b were localized mainly in the cytosol, and only 6% of cellular calpastatins were detected in the membrane fraction. By contrast, more than 30% of the 32P-labeled calpastatins a and b were distributed in the membrane fraction. Thus, the phosphorylation of calpastatin may be involved in regulating the calpain-calpastatin protein kinase system by its subcellular distribution.     

Redescription of Caprogammarus gurjanovae Kudrjaschov & Vassilenko, 1966 (Crustacea: Amphipoda) from Hokkaido,Japan, with notes on the taxonomic status of Caprogammarus

Caprogammarus gurjanovae Kudrjaschov & Vassilenko, 1966 (Amphipoda: Caprellidea: Caprogammaridae) was redescribed based on the materials newly collected off Kushiro and Akkeshi, Hokkaido, Japan, which represents the southernmost record of this genus. Caprogammarus and Caprella share an identical feature, that of having the head and perionite I partially fused. Thus, Caprogammarus is considered to be a member of the suborder Caprellidea.     

The 72-kDa Microtubule-Associated Protein from Porcine Brain

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.     

Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.     

Further fractionation of the glycoprotein families of porcine zona pellucida by anion-exchange HPLC and some characterization of the separated fractions

The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.     

Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells

Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation. Essentially all of the radioactive mannose was released from the protein by treatment with endoglucosaminidase H. The labeled oligosaccharide(s) released by endoglucosaminidase H was isolated and characterized by gel filtration and by treatment with various enzymes. The major oligosaccharide chain on the soybean glucosidase II appeared to be a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2.     

Nδ-benzoyl-l-ornithine and Nδ-benzoyl-l-γ-hydroxyornithine from Vicia pseudo-orobus

Two new non-protein amino acids, N^δ-benzoyl-l-ornithine and N^δ-benzoyl-l-γ-hydroxyornithine have been characterized from the seeds of Vicia pseudo-orobus.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.