Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.     

Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey

Vertebrate lens tissues contain several species of acidic andneutral glycosphingolipids in relatively high amounts. However,the epithelia with capsule from dog and rhesus monkey lenseshad a simpler composition and lower content of glycosphingolipidsthan whole lenses. Gangliosides and neutral glycosphingolipidsin monolayer cultures of lens epithelial cells were also differentfrom those in whole lenses. Although -galactosyl (Gal1-3Ga1-R)or Lewis^x (Galß1-4[Fuc1-3]GlcNAc-R) epitopes werefound in glycosphingolipids from whole lenses, they were notdetected in those from monolayer cultures of dog and rhesusmonkey lens cells. In addition, significant changes in ganglio-seriesgangliosides were induced in monolayer cultures of both cells,where GM3 and GD3 were predominant. Immunofluorescence studyrevealed a characteristic distribution of cell surface gangliosidesin confluent monolayers. These findings suggest that glycosphingolipidsynthesis in lens epithelia is intrinsically different fromthat in cortical and nuclear fibres, and that the expressionof Lewis^x and -galactosyl epitopes in glycosphingolipids appearsto be associated with the differentiation of epithelial cellsto fibres. gangliosides glycosphingolipids lens epithelial cells Lewis^x rhesus monkey.     

Analyses of beta-1 syntrophin, syndecan 2 and gem GTPase as candidates for chicken muscular dystrophy.

Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.     

A compound possessing antitumor and interferon-inducing activities derived from the common antigen (OEP) of Pseudomonas aeruginosa.

OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities. As regards the antitumor activity of the sample, the ED50 value against ascites sarcoma-180 was 1 microgram/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01 microgram/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion. The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material. Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids; the former contained C14:10, C16:0, C16:1, C18:0, and C15:1 acids and the latter, beta-OH C10:0, C12:0, alpha-OH C12:0, beta-OH C12:0, C16:0, and C16:1 acids. The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

A novel difucosylated neutral glycosphingolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus: I. Purification and structural determination of the glycolipid.

A novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fuc alpha 1-3GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-4Glc beta 1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Gal beta 1-4Glc but GlcNAc beta 1-4Glc; it includes a GalNAc beta 1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

Microbial sensor system for nondestructive evaluation of fish meat quality

A microbial sensor system consisting of the bacterium (Alteromonas putrefaciens) immobilized within membranes, a flow cell, an oxygen electrode, peristaltic pumps, a buffer tank, a thermostatically controlled bath and a recorder, was constructed for the nondestructive quality evaluation of bluefin tuna. The chemical compounds on fish meat surfaces which are the indicators of fish meat quality were rapidly determined by using the proposed sensor system. Fish meat quality was determined from the rate of current decrease of the sensor. Good correlations were obtained between fish meat quality and sensor response. One assay could be completed within one minute.     

Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.