A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Purification and Characterization of Cadmium-Binding Protein from Unicelluar Alga Chlorella sorokinian

The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals, and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-binding protein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized. The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfate precipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed by Sephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cadmium binding capacity of the purified protein was 119 μg/mg. The involvement of thiol coordination in metal-ion binding was confirmed by measuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.     

Dietary NaCl supplementation prevents muscle necrosis in a mouse model of Duchenne muscular dystrophy

The mdx mouse is an animal model for Duchenne muscular dystrophy. Mdx mice fed a 12% NaCl diet from birth up to 20 days of age (mdx-Na mice) had an approximately 50% reduction in serum creatine kinase (CK) activity compared with mdx mice fed a standard diet. Most notably, necrotic fibers in tibialis anterior (TA) muscle of mdx-Na mice were reduced by 99% and were similar in control mice. These mdx mice displayed significantly elevated blood Ca2+ and Na+ levels, while the total calcium content of their TA muscle was reduced to the level of control mice. In addition, mdx-Na mice had elevated zinc and magnesium contents in their TA muscle. These results suggest that elevated serum Na+ leads to Ca2+ extrusion from muscle via the Na+/Ca2+ exchanger causing a decrease in intracellular Ca2+ levels and an increase in blood Ca2+ levels. Extracellular Ca2+ and, in addition, Zn2+ and Mg2+ might also contribute to the stabilization of the cell membrane. Other possibilities explaining the surprisingly efficacious beneficial effect of dietary sodium exist and are discussed.     

Structure of anthocyanin from the blue petals of Phacelia campanularia and its blue flower color development

The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be 3-O-(6-O-(4'-O-(6-O-(4'-O-beta-d-glucopyranosyl-(E)-caffeoyl)-beta-d-glucopyranosyl)-(E)-caffeoyl)-beta-d-glucopyranosyl)-5-O-(6-O-malonyl-beta-d-glucopyranosyl)delphinidin. The CD of the blue petals of the phacelia showed a strong negative Cotton effect and that of the suspension of the colored protoplasts was the same, indicating that the chromophores of phacelianin may stack intermolecularly in an anti-clockwise stacking manner in the blue-colored vacuoles. In a weakly acidic aqueous solution, phacelianin displayed the same blue color and negative Cotton effect in CD as those of the petals. However, blue-black colored precipitates gradually formed without metal ions. A very small amount of Al(3+) or Fe(3+) may be required to stabilize the blue solution. Phacelianin may take both an inter- and intramolecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of metal ions. We also isolated a major anthocyanin from the blue petals of Evolvulus pilosus and revised the structure identical to phacelianin.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase

Two essential residues playing critical roles in determining the substrate specificities of cytosolic glutamine synthetase (GS1) have been identified from the alignment of high-affinity (GLN1;1 and GLN1;4) and low-affinity (GLN1;2 and GLN1;3) GS1 isoenzymes in Arabidopsis, and confirmed by site-directed mutagenesis. The results indicated that either K49Q or A174S mutation is sufficient to increase the catalytic efficiencies of GLN1;3 by decreasing its Km values for ammonium. In contrast, replacement of Gln49 and Ser174 by lysine and alanine, respectively, was detrimental to glutamine synthetic activities in GLN1;4. The results suggested that Gln49 and Ser174 in the high-affinity GS1 isoenzymes are interchangeable with Lys49 and Ala174 in the low-affinity variants at the corresponding positions.     

A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls

An auxin analog, 2,4-D, stimulates the activity of endo-1,4-beta-glucanase (EGase) in rice (Oryza sativa L.). The auxin-induced activity from three protein fractions was purified to homogeneity from primary root tissues (based on SDS-PAGE and isoelectric focusing after Coomassie brilliant blue staining). Amino acid sequencing indicated that the 20 N-terminal amino acid sequence of the three proteins was identical, suggesting that these proteins may be cognates of one EGase gene. An internal amino acid sequence of the the rice EGase (LVGGYYDAGDNVK) revealed that this enzyme belongs to glycosyl hydrolase family 9 (GHF9). The major isoform of this rice GHF9 [molecular weight based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): 51,216, isoelectric point (pI): 5.5] specifically hydrolyzed 1,4-beta-glycosyl linkages of carboxymethyl (CM)-cellulose, phosphoric acid-swollen cellulose, 1,3-1,4-beta-glucan, arabinoxylan, xylan, glucomannan, cellooligosaccharides [with a degree of polymerization (DP) >3] and 1,4-beta-xylohexaose, indicating a broader substrate range compared with those of other characterized GHF9 enzymes or EGases from higher plants. Hydrolytic products of two major hemicellulosic polysaccharides in type II cell walls treated with the purified enzyme were profiled using high-performance anion exchange chromatography (HPAEC). The results suggested that endolytic attack by rice EGase is not restricted to either the cellulose-like domain of 1,3-1,4-beta-glucan or the unsubstituted 1,4-beta-xylosyl backbone of arabinoxylan, but results in the release of smaller oligosaccharides (DP <6) from graminaceous hemicelluloses. The comparatively broader substrate range of this EGase with respect to beta-1,4-glycan backbones (glucose and xylose) may partly reflect different roles of gramineous and non-gramineous GHF9 enzymes.