Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

A novel and efficient method for enzymatic synthesis of high purity maltose using moranoline (1-deoxynojirimycin).

A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by beta-amylase [EC 3.2.1.2] from sweet potatoes. The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.