The crystal structure of di-D-fructose anhydride III,produced by inulin D-fructotransferase

Reaction of methyl α-d-glucopyranoside and methyl α-d-mannopyranoside with alkaline hydrogen peroxide and a ferrous salt, at room temperature and below, afforded the corresponding d-glycosiduronic acids. On dehydration, the acids gave the corresponding gamma lactones, with a shift of the pyranoid ring to a furanoid ring. Surprisingly, the glycosidic methyl group was retained during the oxidation reactions and pyranose-furanose interconversions. This retention is rationalized by a mechanism involving formation of a pseudo-acyclic intermediate. Another unexpected reaction was the conversion of slightly moist methyl d-glucopyranosiduronolactone syrup, on standing for 5–6 days at room temperature, into crystalline d-glucofuranurono-6,3-lactone, and of methyl α-d-mannopyranosidurono-6,3-lactone into crystalline d-mannofuranurono-6,3-lactone.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Specific and abundant secretion of a novel hydroxyproline-rich glycoprotein from salt-adapted winged bean cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl₃, but little was secreted in the presence of KCl, LiCl, CaCl₂, MgCl₂, mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp₅) instead of tetrahydroxyproline blocks (Hyp₄) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHuEPO) analogues was developed. The assay took four days, normal mice were used and radioactive compounds were not needed. EPO activity was measured by the increased number of some part of reticulocytes which increased specifically and dose-dependently by the injection of rHuEPO. They were considered to be mostly immature reticulocytes and were counted as the residual particles from blood cells after treatment with a hemolysing reagent. These particles could be counted by conventional automated microcell counters. The assay procedure was simple and easy. The sensitivity, reliability and reproducibility of this method were acceptable for routine in vivo bioassay of rHuEPOs. This method was economical, and can be used instead of the existing bioassays for rHuEPOs.     

Differential diet breadths and species coexistence in leafroller-hunting eumenid wasps

There are, at least, three possible ways in which similar species coexist; resource partitioning, interference competition, and exploitation competition. Here, I investigated which way contributed to the coexistence of leafroller-hunting eumenid wasp species. Resource partitioning and, in addition, differential diet breadths proved to promote species coexistence in this case. First, I analyze the prey records and diet overlap of four eumenid species in a local area. The larger two eumenids hunted similar-sized prey items and had similar potential taxonomic prey uses. But the diet breadth of the subsocial eumenid was much wider than that of the solitary one. As a result, the diet overlap between the two large eumenids decreased. This was because the solitary eumenid attend repeatedly to the same hunting site inhabited by one abundant prey species, while the subsocial one made random hunting. On the other hand, the two medium-sized eumenids partitioned resources according to prey size. Secondly, I related these results to prey choice by several other species of eumenid obtained from literature sources. Ten Japanese common eumenids were divided into four groups according to their prey size. In each of the four groups, 2 to 3 wasp species differentiated the habitat (1 group) or coexisted by means of differential diet breadths (parallel with differential sociality, 2 groups).     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.