Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.     

Crystal structure of LexA: a conformational switch for regulation of self-cleavage

LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.     

A Ran-independent pathway for export of spliced mRNA

All major nuclear export pathways so far examined follow a general paradigm. Specifically, a complex is formed in the nucleus, containing the export cargo, a member of the importin-beta family of transporters and RanGTP. This complex is translocated across the nuclear pore to the cytoplasm, where hydrolysis of the GTP on Ran is stimulated by the GTPase-activating protein RanGAP. The activity of RanGAP is increased by RanBP1, which also promotes disassembly of RanGTP-cargo-transporter complexes. Here we investigate the role of RanGTP in the export of mRNAs generated by splicing. We show that nuclear injection of a Ran mutant (RanT24N) or the normally cytoplasmic RanGAP potently inhibits the export of both tRNA and U1 snRNA, but not of spliced mRNAs. Moreover, nuclear injection of RanGAP together with RanBP1 blocks tRNA export but does not affect mRNA export. These and other data indicate that export of spliced mRNA is the first major cellular transport pathway that is independent of the export co-factor Ran.     

Proteasome activity is required for T lymphocyte aggregation after mitogen activation

The proteasome is a multicatalytic complex of proteases involved in T lymphocyte proliferation and activation through multiple mechanisms. In this study, we investigated its role in lymphocyte aggregation. We found that blocking proteasome activity by a proteasome-specific inhibitor lactacystin (LAC) prevented clustering of T lymphocytes after stimulation with various mitogens. Expression of adhesion molecules ICAM-1 and LFA-1 at cell surfaces of activated T cells was decreased after treatment with LAC. Mechanisms by which the proteasome intervenes in the expression of these adhesion molecules were different. LAC inhibited ICAM-1 expression at the mRNA level, whereas LFA-1 inhibition was probably at a post-translational level. Downregulation of these molecules after proteasome inhibition likely contributes to the observed repression of T cell aggregation. Our results show that the proteasome plays an important role in cell-cell interaction during T cell activation.     

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.     

Tumour necrosis factor-alpha enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alpha involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-alpha on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca(2+) mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiated BK-induced IP accumulation and Ca(2+) mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-alpha and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)), since [3H]BK binding to TSMCs was inhibited by the B(2) selective agonist and antagonist, BK and Hoe 140, but not by the B(1) selective reagents. The enhancing effects of TNF-alpha and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-alpha may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-alpha and PDGF-BB and attenuated the effect of TNF-alpha on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-alpha might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Sequence-dependent flexibility in promoter sequences

The non-neighbor interactions between base-pairs were taken into account to calculate the angular parameters (Omega, rho and tau) describing the orientation of successive base-pair planes and the translation parameters (D(y)) along the long axis of base-pair steps for 36 independent tetramers. A statistical mechanical model was proposed to predict the DNA flexibility that is mainly related to the thermal fluctuations at individual base-pair steps. The DNA flexibility can be described by the root-mean-square deviation of the end-to-end distance of DNA helical structure. The present model was then used to investigate the extreme flexible pattern in prokaryotic and eukaryotic promoter sequences. The results demonstrated several extreme flexible regions related to functionally important elements exist both in prokaryotic promoters and in eukaryotic promoters, DNA flexibility and AT content are highly correlated. The probabilities finding flexibility pattern in promoter sequences were also estimated statistically. The biological implications were discussed briefly.