Structure and expression of cDNA for calphobindin II, a human placental coagulation inhibitor

Calphobindin II, with Mr 73,000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental lambda gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2,361 nucleotides and a 64-nucleotide-long poly(A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.     

Structural studies of fertilization-associated carbohydrate-rich glycoproteins (hyosophorin) isolated from the fertilized and unfertilized eggs of flounder, Paralichthys olivaceus. Presence of a novel penta-antennary N-linked glycan chain in the tandem repeating glycopeptide unit of hyosophorin

New glycoproteins of 100-120 kDa were isolated from the unfertilized eggs of flounder, Paralichthys olivaceus. Compositionally indistinguishable glycopeptides of 6 kDa were also purified from the activated or fertilized eggs. These high and low molecular mass glycoproteins are characterized by high (about 85%) carbohydrate content. Although some heterogeneities exist in the amino acid sequences, the 6-kDa glycopeptides (decapeptides with single large N-linked glycan chains), isolated from the fertilized eggs are the repeating units of the high molecular mass glycoproteins. As judged from several distinctive features the 100-120-kDa glycoproteins are apparently major components of cortical alveoli of flounder eggs and are regarded as members of glycoproteins we have defined under the name of "hyosophorin" (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553). Composition analysis, Smith degradation, hydrazinolysis-nitrous acid deamination, permethylation analysis, and 400-MHz 1H NMR spectroscopy provided evidence for the structure of a novel penta-antennary glycan chain attached to the repeating unit (decapeptide) of the protein core. The structure thus determined is: (Formula: see text). The presence of a unique class of carbohydrate-rich glycoproteins (H-hyosophorin) in the unfertilized eggs, their conversion to the repeating unit (L-hyosophorin) at fertilization, and the finding of a free glycan chain that was formed by scission between the GlcNAc and Asn residues of L-hyosophorin, in the fertilized eggs including embryos of 4-11-h postinsemination, support the view that these molecules may be important in fertilization and subsequent development.     

Fine structure of the dorsal lingual epithelium of the Japanese monkey Macaca fuscata fuscata.

Filiform papillae, which were densely distributed all over the dorsal surface of the lingual body, were crown-shaped, with a central, circular area that sloped in the anterior direction and several branches that surrounded it in a semicircle from the back of the central area. Dome-shaped, fungiform papillae were scattered among these filiform papillae. At the posterior end of the lingual body, there were four circumvallate papillae. Prominent microridges and elevated intercellular borders were widely distributed in the central area of the filiform papillae and the interpapillar region. On the surface of the branches of the filiform papillae, microridges were rarely seen. On the surface of the fungiform papillae, indistinct microridges were observed. Histologically, the dorsal lingual epithelium revealed three different regions: the epithelium on the anterior side of the filiform papillae, the epithelium on the posterior side of the filiform papillae and the interpapillar epithelium. Whereas the basal and suprabasal cells are similar throughout, differences characterize the intermediate and surface layers. Keratohyalin granules appear predominantly in the intermediate layer in the epithelium on the anterior side of filiform papillae. In the epithelium on the posterior side of the filiform papillae, no keratohyalin granules occur and, instead, tonofibrils are prominent. The cells become significantly flattened. In the interpapillar epithelium, no keratohyalin granules are visible, and the tonofilaments occupy almost the entire cytoplasm of most cells in the intermediate and surface layers. The cells are larger in volume in these layers.     

Purification and characterization of a sialidase from Bacteroides fragilis SBT3182.

A sialidase from Bacteroides fragilis SBT3182 was purified 2,240-fold to apparent homogeneity by ammonium sulfate precipitation and sequential chromatographies on DEAE-Toyopearl 650M, Hydroxyapatite, MonoS and Superose6 columns. The N-terminal amino acid sequence of this sialidase, Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-, was determined. Substrate specificity of this enzyme using a variety of sialoglycoconjugates showed a 1.5- and 2.2-fold preference for sialyl alpha 2-8 linkages when compared with alpha 2-3 and alpha 2-6 bound sialic acids, respectively. The native sialidase had a molecular weight of 165kDa, as determined by Superose6 gel filtration chromatography and consisted of three subunits each of 55kDa by SDS-polyacrylamide gel electrophoresis. This enzyme had optimal activity at pH6.1 with colominic acid as substrate.     

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When -adrenoceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.     

Concanamycin 4-B: A Potent Inhibitor of Vacuolar pH Regulation in Chara Cells

Concanamycin 4-B, a macrolide antibiotic with an 18-membered lactone ring, is known as a specific inhibitor of the vacuolar type of H⁺-ATPase, as is bafilomycin A₁. The drug was tested for its effect on regulation of the vacuolar pH (pH_v) of internodal cells of a fresh water characean alga, Chara corallina, under normal conditions and under salt stress. The pH_v was measured either on isolated vacuolar sap with a conventional pH electrode or directly by inserting a pH-sensitive glass microelectrode into the vacuole. Proton-pumping into tonoplast vesicles was almost completely inhibited by concanamycin 4-B at 1 nM. Concanamycin 4-B at 1 μM significantly increased pH_v while bafilomycin A₁ was ineffective when applied at 1 μM. Concanamycin 4-B did not affect pH_v when applied at 0.1 μM and increasing the concentration to 10 μM did not amplify the degree of alkalization. Concanamycin 4-B also inhibited pH_v regulation under NaCl stress. When Chara cells were treated with 100 mM NaCl, pH_v promptly increased and then recovered to the original level. The reacidification was completely inhibited by concanamycin 4-B (1 μM), suggesting that the reacidification was achieved by the H⁺-ATPase of the tonoplast.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Light-Induced Changes in Cytosolic pH in Leaf Cells of Egeria densa: Measurements with pH-Sensitive Microelectrodes

Light-induced changes of cytosolic pH (pH_c) and the plasmalemmapotential (E_m) in dark-adapted leaf cells of the aquatic plant,Egeria densa were measured simultaneously with double-barreledpH-sensitive microelectrodes. Upon illumination, pH_c increasedtransiently and then decreased to a level that was lower thanthe original value, while the plasmalemma was greatly hyperpolarizedafter an initial small depolarization. DCMU inhibited the light-inducedchanges in both pH_c and E_m. DCMU acted without directly inhibitingthe electrogenic proton pump in the plasmalemma since a decreasein pH_c caused by treatment with butyrate (H⁺-loading) hyperpolarizedthe plasmalemma in DCMU-pretreated cells. N.N-Dicyclohexylcarbodiimide(DCCD) also inhibited the light-induced changes in both pH_cand E_m. This result may be explained by direct inhibition ofthe proton pump in the plasmalemma by DCCD since the decreasein pH_c caused by butyrate did not induce membrane hyperpolarizationin DCCD-treated leaf cells. Fusicoccin induced membrane hyperpolarizationand slight acidification of the cytosol. DCCD inhibited thefusicoccin-induced changes in both pH_c and E_m. The mechanismof the light-induced changes in pH_c is discussed in relationto activities of the proton pump in the plasmalemma and photosynthesis. (Received January 10, 1994; Accepted June 9, 1994)     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae.

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.