Effect of outer sphere anions on the structure and color of nitrosylpentaamminechromium complex

Three kinds of crystalline compounds containing the nitrosylpentaamminechromium complexes [Cr(NO)(NH₃)₅]²⁺(A) were obtained: chloride ACl₂ (red-orange), chloride perchlorate ACl(ClO₄) (brown), and perchlorate A(ClO₄)₂ (green). The cause of the color change of the complex A with the change of outer sphere anions was sought using X-ray structural data of ACl₂, ACl(ClO₄), and A(ClO₄)₂. Crystal data: ACl₂, orthorhombic, space group Cmcm, a=10.0236 (9) Å, b=9.098 (3) Å, c=10.357(1) Å, V=944.5 (5) ų, Z=4; ACl(ClO₄), tetragonal, space group P4/nmm, a=7.6986 (8) Å, c=9.9566(8) Å, V=590.1 (1) Å^3,Z=2; A(ClO₄)₂, orthorhombic, space group Pnma, a=15.760 (2) Å, b=11.480(2) Å, c=7.920 (2) Å, V=1432.9 (4) ų, Z=4. The complex cation in ACl₂ has a distorted octahedral structure with a linear CrNO moiety. The short CrN (nitrosyl) distance of 1.692 (7) Å indicates the presence of multiple bonding between the chromium atom and the nitrogen atom in the nitrosyl group. The interatomic distances and angles within the complex cations hardly change with the change of the counter anions, while the distances between the complex cations in each crystal increase in the order ACl₂<ACl(ClO₄)<A(ClO₄)₂. The bulky perchlorate anions seems to separate the complex cations, while smaller chloride anions are not large enough to separate them. The distance (3.213(5) Å) between O(NO) and N(NH₃ in the adjacent complex cation) is rather short in the crystal of ACl₂, and there are six hydrogen bonds, where the NO group is surrounded by four NH₃ ligands. The distance (4.002(5) Å) between O(NO) and N(NH₃) is much longer in the crystal of A(ClO₄)₂, indicating the presence of no hydrogen bonding. In the crystal of ACl(ClO₄) the distance (3.452(4) Å) between O(NO) and N(NH₃) is in between those of ACl₂ and A(ClO₄)₂. The presence of hydrogen bonding between O(NO) and N(NH₃ in the adjacent complex cation) seems to cause the color change with the change of outer sphere anions.     

Expression of programmed death 1 ligands by murine T cells and APC

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.     

Relationship between conidial enzymes and germination of the apple blue mold,Penicillium expansum

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding

A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.     

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.     

Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis

Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites. We have determined the complete nucleotide sequence of the linear chromosome of Streptomyces avermitilis. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome contains 9,025,608 bases (average GC content, 70.7%) and encodes at least 7,574 potential open reading frames (ORFs). Thirty-five percent of the ORFs (2,664) constitute 721 paralogous families. Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. Comparison with Streptomyces coelicolor A3(2) revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes but showed perfectly asymmetric structure at the oriC center. In contrast, the terminal regions were not conserved and preferentially contained nonessential genes.     

Electron microscopic evidence for the thick filament interconnections associated with the catch state in the anterior byssal retractor muscle of Mytilus edulis

The anterior byssal retractor muscle (ABRM) of a bivalve mollusc Mytilus edulis is known to exhibit catch state, i.e. a prolonged tonic contraction maintained with very little energy expenditure. Two different hypotheses have been put forward concerning the catch state; one assumes actin-myosin linkages between the thick and thin filaments that dissociate extremely slowly (linkage hypothesis), while the other postulates a load-bearing structure other than actin-myosin linkages (parallel hypothesis). We explored the possible load-bearing structure responsible for the catch state by examining the arrangement of the thick and thin filaments within the ABRM fibers, using techniques of quick freezing and freeze substitution. No thick filament aggregation was observed in the cross-section of the fibers quickly frozen not only in the relaxed and actively contracting states but also in the catch state. The thick filaments were, however, occasionally interconnected with each other either directly or by distinct projections in all the three states studied. The proportion of the interconnected thick filaments relative to the total thick filaments in a given cross-sectional area was much larger in the catch state than in the relaxed and actively contracting states, providing evidence that the thick filament interconnection is responsible for the catch state.     

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.