Nitric oxide down-regulates MKP-3 mRNA levels: involvement in endothelial cell protection from apoptosis

MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor alpha (TNFalpha) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFalpha-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFalpha were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of the S-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA.     

Enhanced epinephrine-induced platelet aggregation in individuals carrying the G protein beta3 subunit 825T allele

The 825T allele of a common C825T polymorphism in the gene encoding the beta3 subunit of heterotrimeric G proteins is associated with enhanced activation of pertussis toxin (PTX)-sensitive G proteins. We investigated responses of human platelets upon stimulation with epinephrine, which activates PTX-sensitive G proteins, and with agonists which activate additionally, or exclusively PTX-insensitive pathways. Slopes and maximum of the secondary aggregation were significantly enhanced in platelets from 825T allele carriers after epinephrine, and after combined epinephrine/ADP. This effect was more pronounced after inhibition of the cyclooxygenase-2 pathway by acetylsalicylic acid. This phenomenon appeared independent of platelet secretion, or inhibition of the adenylyl cyclase.     

Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions

The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.     

Structure and interfacial properties of chicken apolipoprotein A-IV

To gain insight into the evolution and function of apolipoprotein A-IV (apoA-IV) we compared structural and interfacial properties of chicken apoA-IV, human apoA-IV, and a recombinant human apoA-IV truncation mutant lacking the carboxyl terminus. Circular dichroism thermal denaturation studies revealed that the thermodynamic stability of the alpha-helical structure in chicken apoA-IV (DeltaH = 71.0 kcal/mol) was greater than that of human apoA-IV (63.6 kcal/mol), but similar to that of human apoA-I (73.1 kcal/mol). Fluorescence chemical denaturation studies revealed a multiphasic red shift with a 65% increase in relative quantum yield that preceded loss of alpha-helical structure, a phenomenon previously noted for human apoA-IV. The elastic modulus of chicken apoA-IV at the air/water interface was 13.7 mN/m, versus 21.7 mN/m for human apoA-IV and 7.6 mN/m for apoA-I. The interfacial exclusion pressure of chicken apoA-IV for phospholipid monolayers was 31.1 mN/m, versus 33.0 mN/m for human A-I and 28.5 mN/m for apoA-IV.We conclude that the secondary structural features of chicken apoA-IV more closely resemble those of human apoA-I, which may reflect the evolution of apoA-IV by intraexonic duplication of the apoA-I gene. However, the interfacial properties of chicken apoA-IV are intermediate between those of human apoA-I and apoA-IV, which suggests that chicken apoA-IV may represent an ancestral prototype of mammalian apoA-IV, which subsequently underwent further structural change as an evolutionary response to the requisites of mammalian lipoprotein metabolism.     

A dimeric DNA interface stabilized by stacked A.(G.G.G.G).A hexads and coordinated monovalent cations

We report on the identification of an A.(G.G.G.G).A hexad pairing alignment which involves recognition of the exposed minor groove of opposing guanines within a G.G.G.G tetrad through sheared G.A mismatch formation. This unexpected hexad pairing alignment was identified for the d(G-G-A-G-G-A-G) sequence in 150 mM Na(+) (or K(+)) cation solution where four symmetry-related strands align into a novel dimeric motif. Each symmetric half of the dimeric "hexad" motif is composed of two strands and contains a stacked array of an A.(G.G.G.G).A hexad, a G.G.G.G tetrad, and an A.A mismatch. Each strand in the hexad motif contains two successive turns, that together define an S-shaped double chain reversal fold, which connects the two G-G steps aligned parallel to each other along adjacent edges of the quadruplex. Our studies also establish a novel structural transition for the d(G-G-A-G-G-A-N) sequence, N=T and G, from an "arrowhead" motif stabilized through cross-strand stacking and mismatch formation in 10 mM Na(+) solution (reported previously), to a dimeric hexad motif stabilized by extensive inter-subunit stacking of symmetry-related A.(G.G.G.G).A hexads in 150 mM Na(+) solution. Potential monovalent cation binding sites within the arrowhead and hexad motifs have been probed by a combination of Brownian dynamics and unconstrained molecular dynamics calculations. We could not identify stable monovalent cation-binding sites in the low salt arrowhead motif. By contrast, five electronegative pockets were identified in the moderate salt dimeric hexad motif. Three of these are involved in cation binding sites sandwiched between G.G.G. G tetrad planes and two others, are involved in water-mediated cation binding sites spanning the unoccupied grooves associated with the adjacent stacked A.(G.G.G.G).A hexads. Our demonstration of A.(G. G.G.G).A hexad formation opens opportunities for the design of adenine-rich G-quadruplex-interacting oligomers that could potentially target base edges of stacked G.G.G.G tetrads. Such an approach could complement current efforts to design groove-binding and intercalating ligands that target G-quadruplexes in attempts designed to block the activity of the enzyme telomerase.     

Effect of chronic excess of tumour necrosis factor-alpha on contractile proteins in rat skeletal muscle

The effect of chronic tumour necrosis factor-alpha (TNF-alpha) treatment on the synthesis of specific myofibrillar proteins such as heavy chain myosin, light chain myosin and G-actin in rat diaphragm were evaluated. Muscles (diaphragm) from control and experimental groups (TNF-alpha i.v. at 50 microg/kg body wt for 5 days) were incubated in the presence of 35S-methionine for 2 h. Myofibrillar protein extracts were prepared and protein was electrophoresed on sodium dodecyl sulphate-polyacrylamide gels. Heavy chain myosin, light chain myosin and G-actin were identified by Western blot analysis using specific monoclonal antibodies. Polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis revealed two types of heavy chain myosin (206 and 212 kD), all four types of light chain myosin (15, 16.5, 18 and 20 kD) and a single type of G-actin (42 kD). Chronic TNF-alpha treatment produced a significant decline in the synthesis of all types of myofibrillar proteins, namely heavy chain myosin, light chain myosin and G-actin. TNF-alpha impaired peptide-chain initiation in diaphragm muscle which was reversed by the branched-chain amino acids (BCAA) therapy of TNF-alpha treated rats. These findings indicate a significant role for TNF-alpha in the translational regulation of protein synthesis in skeletal muscle.     

Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.     

Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine arctic sediments

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54. 8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1) day(-1)), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

RNA bulges as architectural and recognition motifs

RNA bulges constitute versatile structural motifs in the assembly of RNA architectures. Three-dimensional structures of RNA molecules and their complexes reveal the role of bulges in RNA architectures and illustrate the molecular mechanisms by which they confer intramolecular interactions and intermolecular recognition.     

Reaktivierung von UV-Schäden in Salmonella typhimurium

Summary The uv- and x-ray sensitivity for colony formation of Salmonella typhimurium has been compared. If the comparation is done on the basis of equal sensitivity of the bacterial gene responsible for host reactivation of uv-damaged P22, the uv-sensitivity of colony formation is only 1/4 of the sensitivity to x-ray irradiation.It is concluded, that 3/4 of the uv-damage to colony formation in Salmonella typhimurium can be reactivated by the cell itself. This reactivable sector of uv-damage in the bacterial cell is the same as the sector of hostreactivable uv-damages in the phage P22 which is able to grow and to be hostreactivated in Salmonella typhimurium.