Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure

The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2). Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No. 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure. Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides. The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base. Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S. and Uesugi, S. (1991) FEBS Lett. 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.     

Linkage between alpha(1) adrenergic receptor and the Jak/STAT signaling pathway in vascular smooth muscle cells

The Jak/STAT pathway is activated following stimulation of the type I angiotensin II receptor. To examine whether this pathway is shared among other G-protein-coupled receptors, we studied the linkage between the alpha(1) adrenergic receptor and this pathway. The alpha(1) agonist phenylephrine induced tyrosine phosphorylation of Jak2, Tyk2, and STAT1 in vascular smooth muscle cells. The phosphorylation of Jak2 was prevented by the alpha(1) receptor antagonists prazosin and chloroethylclonidine, but not by WB4101, and that of STAT1 was inhibited by prazosin and the Jak2 inhibitor AG490. After stimulation with phenylephrine, Jak2 and STAT1 were found to associate with alpha(1B) receptor. Phenylephrine stimulated the DNA binding activity of STAT1. Protein synthesis promoted by phenylephrine was inhibited by prazosin, AG490, and the introduction of a decoy oligonucleotide for STAT1. These results suggested that alpha(1) receptor is linked to the Jak/STAT pathway and that this pathway mediates alpha(1) agonist-induced smooth muscle hypertrophy.     

The solution structures of two soybean calmodulin isoforms provide a structural basis for their selective target activation properties

The intracellular calcium ion is one of the most important secondary messengers in eukaryotic cells. Ca(2+) signals are translated into physiological responses by EF-hand calcium-binding proteins such as calmodulin (CaM). Multiple CaM isoforms occur in plant cells, whereas only a single CaM protein is found in animals. Soybean CaM isoform 1 (sCaM1) shares 90% amino acid sequence identity with animal CaM (aCaM), whereas sCaM4 is only 78% identical. These two sCaM isoforms have distinct target-enzyme activation properties and physiological functions. sCaM4 is highly expressed during the self-defense reaction of the plant and activates the enzyme nitric-oxide synthase (NOS), whereas sCaM1 is incapable of activating NOS. The mechanism of selective target activation by plant CaM isoforms is poorly understood. We have determined high resolution NMR solution structures of Ca(2+)-sCaM1 and -sCaM4. These were compared with previously determined Ca(2+)-aCaM structures. For the N-lobe of the protein, the solution structures of Ca(2+)-sCaM1, -sCaM4, and -aCaM all closely resemble each other. However, despite the high sequence identity with aCaM, the C-lobe of Ca(2+)-sCaM1 has a more open conformation and consequently a larger hydrophobic target-protein binding pocket than Ca(2+)-aCaM or -sCaM4, the presence of which was further confirmed through biophysical measurements. The single Val-144 --> Met substitution in the C-lobe of Ca(2+)-sCaM1, which restores its ability to activate NOS, alters the structure of the C-lobe to a more closed conformation resembling Ca(2+)-aCaM and -sCaM4. The relationships between the structural differences in the two Ca(2+)-sCaM isoforms and their selective target activation properties are discussed.     

1,25-Dihydroxyvitamin D3 regulates proliferation of activated T-lymphocyte subsets

The biologically active vitamin D metabolite, 1,25-(OH) 2D3 suppressed phytohaemagglutinin (PHA)-induced lymphocyte proliferation dose-dependently (0.1 nM-100 nM), and decreased the OKT4+/OKT8+ ratio and transferrin-receptor-positive (OKT9+) cells. A possible parallelism between expression of 1,25-(OH) 2D3 receptors and interleukin 2 (IL2)-receptors recognized by anti-Tac antibody was not confirmed in this study. However, the addition of exogenous IL2 abolished the inhibitory effects of 1,25-(OH) 2D3 on PHA-stimulated T-cell proliferation, and the decrease of OKT4+ and OKT9+ T-cell in this population. Among various vitamin D3 analogues examined, 1,25-(OH) 2D3 was the most potent anti-proliferative effect, followed in order by 1,24S-(OH) 2D3, 1 alpha OH D3, 25 OH D3 and 24,25-(OH) 2D3.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity

Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at > 2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.