Biochemical and functional characterization of a eukaryotic-type protein kinase,SpkB, in the cyanobacterium,Synechocystis sp. PCC 6803

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+). Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.     

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity,increases expression and propagation of BLV in vitro but not in vivo

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.     

Responsiveness of insulin-induced cardiac sympathetic nerve activation associates with blood pressure regulation in diabetics

To quantitatively evaluate the effect of insulin on cardiac sympathetic nerve activity (SNA) and analyze clinical factors associated with insulin sensitivity for the regulation of SNA in diabetics, 29 Japanese type 2 diabetics without neuropathy were recruited. A 2-h control study and a 2-h hyperinsulinemic euglycemic glucose clamp study were performed. From the power spectral analysis of R-R intervals on ECG during both studies, two major components, the low-frequency (LF) and the high-frequency component (HF), were obtained. Then %LF was calculated as LF/(LF +HF), and the ratio of the average %LF during the last 30 min of the clamp or the control to the average %LF for the entire time for clamp or control (R-%LF) was used as a marker of changes in SNA. R-%LF was significantly higher during the clamp than in the control (1.07 +/- 0.04 vs. 1.03 +/- 0.03, P < 0.05). High responders (individual R-%LF during clamp > or = mean + 2SD in control) showed a higher basal mean blood pressure (BP) before the clamp (89 +/- 3 vs. 82 +/- 2, P < 0.03) but not a higher glucose infusion rate (GIR) compared with low responders (相似文献   

Phosphate metabolites in muscular contraction caused by magnetic stimulation

The metabolism of high energy phosphates during muscular contraction due to direct electrical stimulation, indirect stimulation via nerve excitation, and magnetic stimulation was studied in isolated muscles (frog sartorius muscles) by (31)P nuclear magnetic resonance ((31)P-NMR). Twitch amplitudes elicited by each stimulus were measured alternatively at 3 mm displacement loading and 5 g weight. Both the creatine/inorganic phosphate (PCr/Pi) and pH changes were more marked in direct electrical stimulation than in magnetic stimulation. The muscular contraction caused by magnetic stimulation showed less fatigue than that caused by direct electrical muscular stimulation.     

Modulation of procarboxypeptidase R (ProCPR) activation by complementary peptides to thrombomodulin

We designed complementary peptides (C-peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsin-like enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200-fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C-peptides which recognize the T-binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T-TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 microM reduced the activation of proCPR to the level obtained by T alone.     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

Stereoselective C-glycosidation of unprotected D-glycals with trimethylsilyl cyanide

Unprotected glycals reacted with trimethylsilyl cyanide in the presence of a catalytic amount of a palladium compound to yield the 2,3-unsaturated glycosyl cyanides in high yield and in alpha-selectivity.