Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Study of amphibian lipids. I. Characterization of monoglycosylceramides from the skin of Rana nigromaculata (Japanese pond frog).

1. Monoglycosylceramide was isolated from the skin of Rana nigromaculata (Japanese pond frog), and further fractionated into three subgroups (Fraction I, Fraction II and Fraction III) by borate-impregnated Florisil column chromatography. 2. Fraction I and Fraction II contained mainly glucose as their hexose components, while Fraction III contained galactose. 3. Major long chain bases of Fraction I and Fraction III were D-erythro-1, 3-dihydroxy-2-amino-4-trans-octadecene (4-sphingenine) and D-erythro-1, 3-dihydroxy-2-aminooctadecane (sphinganine), whereas those of Fraction II were D-ribo-1, 3, 4-trihydroxy-2-aminooctadecane (4D-hydroxysphinganine) and 1, 3, 4-trihydroxy-2-aminoeicosane (C20 homologues of 4D-hydroxysphinganine). This is the first evidence of the presence of trihydroxy base-containing glycolipids in the skin of vertebrates. 4. All three subgroups of monoglycosylceramide contained both hydroxy and nonhydroxy fatty acids ranging from C14 and C26. Saturated fatty acids represented more than 90% of the total. Some differences of the fatty acid composition in the three subgroups were also observed.     

An altered adenylate cyclase in cdc35-1 cell division cycle mutant of yeast

Adenylate cyclase activity was studied in Saccharomyces cerevisiae's cell division cycle (cdc) mutant 35-1. The temperature sensitive mutant cdc35-1 was previously mapped as an allele of cyr, the adenylate cyclase gene. However, the adenylate cyclase activities of membranes prepared from cdc35-1 were not thermosensitive. The adenylate cyclase activity of cdc35-1 was found to have an altered Mn2+ dependency and did not respond to Gpp(NH)p stimulation. These results suggest that cdc35-1 mutation may not be at the catalytic site but at a site where adenylate cyclase interacts with regulatory proteins.     

Further studies on the smooth-surfaced membranes isolated from rat brain

The light and heavy smooth-surfaced membranes (LSM and HSM), which had densities corresponding to 1.08 M and 1.28 M sucrose, respectively, were isolated from rat brain and some of their biochemical properties were investigated. Both LSM and HSM showed high Na+,K+-ATPase activity and, in particular, in HSM the activity was four times (21.55 mumol/mg protein/h) higher than that of the brain homogenate. High 2',3'-cyclic nucleotide 3'-phosphodiesterase activity (293.4 mumol/mg protein/h) was characteristic of LSM. 5'-Nucleotidase and acetylcholinesterase activities were also higher in LSM than in HSM. SDS-polyacrylamide gel electrophoresis showed that LSM and HSM had many protein component and that low molecular weight proteins such as proteolipid protein and basic protein were almost absent, in contrast with myelin and myelin-like membrane. GM1 ganglioside constituted the major class of total ganglioside in both LSM and HSM. These biochemical findings suggested that LSM is a membrane that has not previously been described, or a membrane fraction related to the oligodendroglial plasma membrane.     

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides

A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)^?1 min^?1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking.     

The effect of an in vivo-injected thiol protease inhibitor, E-64-c, on the calcium-induced degeneration of myofilaments

The role of intracellular calcium-dependent proteinase(s) has been investigated in intact rat muscle. When calcium ions were introduced into intact muscle in vitro with ionophore A23187, Z-line loss and concomitant release of alpha-actinin into the medium were observed. The calcium-induced release of alpha-actinin was not diminished in the muscle with in vivo-injection of a thiol protease inhibitor, E-64-c. Intramuscular concentrations of E-64-c were also measured after pulse labeling with [3H]E-64-c followed by subcellular fractionation. Most of the inhibitor was localized in the cytosol, not in the lysosome. Therefore, we conclude that cytosolic as well as lysosomal proteinases in muscle are not inhibited by the in vivo labeling of the protease inhibitor (10 mg/kg).     

Novel epoxysuccinyl peptides Selective inhibitors of cathepsin B, in vitro

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-isoleucyl-L-proline (compound CA-030) and N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline (compound CA-074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n-propylamide group in the oxirane ring were necessary, the ethyl ester group or the n-propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.     

Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K₁ values for cathepsin B were about 2–5 nM, like that of E-64-c, whereas their initial K₁ values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.     

Purification of aromatic L-amino acid decarboxylase from bovine brain with a monoclonal antibody.

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5'-phosphate as a cofactor, both enzymes were similar.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.