Acetolysis of Leuconostoc mesenteroides NRRL B-1299 Dextran. Isolation and characterization of tetrasacharrides containing secondary linkages from the borate-insoluble fraction

Fractionation of the deacetylated acetolyzate of the borate-insoluble fraction of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 gave five tetrasaccharide fractions, isolated after chromatography on charcoal—Celite, paper chromatography, and paper electrophoresis. Examination of partial acid hydrolyzates of the tetrasaccharide fractions and their corresponding alditols, the relation between the logarithm of their partition functions (α') and molecular size, and methylation studies, showed them to be (a) 2³-α-d-glucosyl-nigerotriose (1), (b) a mixture of 6-α-nigerotriosyl-d-glucose (2) and 6¹-α-d-glucosyl-nigerotriose (3) and/or 6²-α-d-glucosyl-nigerotriose (4), (c) a mixture of 2¹-α-nigerosyl-isomaltose (5) and 3²-α-isomaltosyl-kojibiose (6) and/or 6²-α-nigerosyl-kojibiose (7), (d) 2-α-nigerotriosyl-d-glucose (8) and (e) nigerotetraose (9).     

Increase in liver glucose transporter mRNA levels during rat liver regeneration

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.     

Transport of acid phosphatase to lysosomes does not involve passage through the cell surface

In foregoing studies, we reported that LGP107, a major lysosomal membrane glycoprotein in the rat liver, distributes in and circulates continuously throughout the endocytic membrane system (endosomes, lysosomes and plasma membrane), in hepatocytes (1,2). In the present study we examined whether acid phosphatase (APase), an enzyme that is transported to lysosomes as a transmembrane protein, passes through the cell surface during intracellular transport, because transport of newly synthesized APase to lysosomes involves the passage of endosomes containing a ligand which is internalized via receptors on the cell surface and is finally dispatched to lysosomes for degradation (3). When localization of APase in rat hepatocytes was investigated by immunoelectron microscopy, APase was found to be localized in lysosomes and endosomes, but not in coated pits on the cell surface, which are positive for LGP107, and from which antibodies for LGP107 are internalized. Further, unlike LGP107, newly synthesized APase was not detected in plasma membranes isolated from livers of rats given [35S]methionine, and when cultured hepatocytes were exposed to 125I-labeled anti APase IgG at 37 degrees C, there was no transfer of the antibody to lysosomes even after 24 h incubation. Therefore, these results indicate that intracellular movement of APase does not involve cell surface passage in rat hepatocytes, and clearly differs from the recent report that human APase is transported to lysosomes via the cell surface in BHK cells transfected with its cDNA (4).     

Conversion of big endothelin-1 by membrane-bound metalloendopeptidase in cultured bovine endothelial cells

We propose a candidate for the "putative" endothelin (ET) converting enzyme in the cultured endothelial cells (ECs) of bovine carotid artery. The enzyme is membrane-bound, soluble in 0.5% Triton X-100, and capable of converting human big ET-1 to ET-1 by a specific cleavage between Trp21 and Val22. The conversion reached 90% after a 5-hr incubation in the presence of DFP, PCMS and pepstatin A, but it was inhibited by EDTA, omicron-phenanthroline or phosphoramidon. The enzyme is very sensitive to pH, and active only between pH 6.6 and pH 7.6. Conversion of big ET-3 by this enzyme was only 1/9 that of big ET-1. From these results, ET-1 converting enzyme in the bovine EC is most likely to be a membrane-bound, neutral metalloendopeptidase, which is much less susceptible to big ET-3.     

(Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K(+)-site directed probe for Na+/K(+)-ATPase

Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na+/K(+)-ATPase activity. This inactivation was prevented by K+ and its congeners. In this study, we examined the possibility of Ca2+ or ethylenediamine as a probe of the K+ occlusion center of Na+/K(+)-ATPase. The inactivation by AU-1421 was prevented by Ca2+ with an apparent high affinity (approximately 0.1 mM). Ca2+ protection was cancelled by high concentrations of ATP, ADP or Mg2+. Ca2+ and K+ were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C12E8 solubilization of the enzyme, suggesting that ethylenediamine, like K+, stabilized the enzyme. However, an apparent affinity of ethylenediamine (approximately 1.4 mM) was one order of magnitude lower than that of K+ (approximately 0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K+ binding. From these results we concluded that ethylenediamine does not act like K+ or Ca2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E2 (K(+)-bound form).     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain.

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.     

RFLP- and physical mapping of resistance gene homologues in rice (O. sativa) and Barley (H. vulgare)

 The deduced peptide sequences of 25 gene fragments of NBS-LRR resistance (R) gene homologues from rice and barley and of characterized R genes were compared, revealing a string of six conserved motifs. Mapping of the R-gene candidates in rice showed linkage to genes conferring race-specific resistance to rice blast (Pi-k, Pi-f and Pi-1) and bacterial blight disease (Xa-1, Xa-3 and Xa-4), in barley to powdery mildew (Mla) and the rust fungus (Rpg1). In rice four mixed clusters were detected, each harboring at least two highly dissimilar NBS-LRR genes. A YAC-contig was established for one of these mixed clusters. YAC fragmentation experiments revealed the presence of at least five NBS-LRR genes within 200 kb in head-to-tail orientation. Received: 24 July 1998 / Accepted: 14 August 1998     

Reconstituted HDL containing human apolipoprotein A-1 reduces VCAM-1 expression and neointima formation following periadventitial cuff-induced carotid injury in apoE null mice.

Arterial injury triggers an inflammatory response in part mediated by induction of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and is implicated in neointimal thickening. Since HDL is known to reduce cytokine-activated VCAM-1 expression, we tested the hypothesis that VCAM-1 expression and neontimal thickening following arterial injury are inhibited by reconstituted human HDL containing plasma-derived apoA-1 (rHDL). We used the carotid cuff injury in apoE (-/-) mice fed high cholesterol. Mice received rHDL (40 mg/kg) intravenously every other day for 3 weeks. Compared to control, rHDL treatment inhibited neointima formation (0. 008 +/- 0.004 mm(2) vs. 0.037 +/- 0.019 mm(2); P < 0.01) 21 days after injury, reduced VCAM-1 expression, and decreased monocyte/macrophage infiltration as assessed by histomorphometric analysis within the first week after injury. These changes occurred without any effect on plasma total and HDL cholesterol levels as well as the arterial tissue cholesterol levels. rHDL treatment also reduced the formation of modified lipoprotein in the arterial wall compared to control within the first week after injury. This finding suggests an antioxidant effect of rHDL associated with reduced VCAM-1 expression and neointimal formation after arterial injury.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.