Amidase-like activity of calpain I and calpain II on substance P and its related peptides

Porcine calpains (Ca2+-dependent cysteine proteinases) I and II, which had been purified each to a homogeneous state, were found to hydrolyze specifically carboxyl-terminal amide of substance P and several other biologically active peptidyl amides. This amidase-like activity was demonstrated both by determining released ammonia and by separating products on high-performance liquid chromatography followed by amino acid analysis. The calpain-catalyzed deamidation of substance P occurred exclusively at the carboxyl-terminal amide, leaving the side-chain glutamine intact. Enkepharinamide and MSH-release inhibiting factor were scarcely deamidated. Calpains I and II showed similar specificities for these amide substances and similar profiles of inhibitions by various protease inhibitors, but distinctly different Ca2+ requirements. The specificity constants, kcat/Km, for substance P were found to be three to four orders of magnitude higher than those for the synthetic substrates.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.     

Demonstration of structural polymorphism among MB3 light chains by two-dimensional gel electrophoresis

The heavy and light chain subunits of MB3 molecules were isolated from KT2 (DKT2, DR4, MB3 homozygous), ER (Dw4, DR4, MB3 homozygous), JMe (Dw5, DR5, MB3 homozygous), EBV-Sh (DSh, DRw6.2, MB3 homozygous), and EBV-Ky (DKy, DRw9, MB3 homozygous) cells and were compared with one another by two-dimensional gel electrophoresis. The MB3 light chains from KT2, ER, and EBV-Ky cells were clearly different in terms of their isoelectric points, whereas those from ER, JMe, and EBV-Sh cells were indistinguishable. No differences in charge or m.w. were noted for the MB3 heavy chains from the five cell lines. Thus, three out of the five MB3-positive, D/DR-disparate cell lines were found to express structurally distinct MB3 molecules, demonstrating that MB3 is a public serologic specificity shared by at least three structurally distinct MB (human I-A-like) molecules. Because the DR light chain subunits isolated from EBV-Wa, KT2, ER, JMe, EBV-Sh, and EBV-Ky cells differed from one another in their isoelectric points, the DR light chains were apparently more polymorphic than the MB3 light chains.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

O3 Tolerance and the Ascorbate-Dependent H2O2 Decomposing System in Chloroplasts

The relationship between O₃ tolerance and the chloroplast H₂O₂scavenging system (PS I     

Structures of sugar chains of the third component of human complement

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.     

Neighboring proteins in rat liver 60 S ribosomal subunits disulfide-linked by hydrogen peroxide oxidation or cross-linked with dithiobis(succinimidyl propionate)

Neighboring proteins in rat liver 60 S ribosomal subunits were investigated by two kinds of cross-linking techniques: treatment of 60 S subunits with 1) hydrogen peroxide, which promotes the formation of protein-protein disulfide linkages and 2) a disulfide-bridged bifunctional reagent dithiobis(succinimidyl propionate). The cross-linked protein complexes formed were separated by two-dimensional polyacrylamide gel electrophoresis in a basic-sodium dodecyl sulfate gel system under nonreducing conditions. Each complex in the gel was labeled with 125I and extracted under reducing conditions. The protein components of the complex were analyzed by two kinds of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Closely neighboring pairs disulfide-linked by hydrogen peroxide were identified as L4-L6, L4-L29, L6-L29, L18a-L29, and L29-L32; more distant pairs cross-linked with dithiobis(succinimidyl propionate) were identified as L3-L5, L3-L24, L3-L37a, L4-L14, L4-L18a, L5-L10, L5-L11, L7/L7a-L27, L7/L7a-L36, L13-L35, and L13a-L14.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.