A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Complementation of Escherichia coli ubiF mutation by Caenorhabditis elegans CLK-1, a product of the longevity gene of the nematode worm

Caenorhabditis elegans CLK-1 was identified from long-lived mutant worms, and is believed to be involved in ubiquinone biosynthesis. The protein belongs to the eukaryotic CLK-1/Coq7p family, which is also similar to the bacterial Coq7 family, that hydroxylates demethoxyubiquinone, resulting in the formation of hydroxyubiquinone, a precursor of ubiquinone. In Escherichia coli, the corresponding reaction is catalyzed by UbiF, a member of a distinct class of hydroxylase. Although previous studies suggested that the eukaryotic CLK-1/Coq7 family is a hydroxylase of demethoxyubiquinone, there was no direct evidence to show the enzymatic activity of the eukaryotic CLK-1/Coq7 family. Here we show that the plasmid encoding C. elegans CLK-1 supported aerobic respiration on a non-fermentable carbon source of E. coli ubiF mutant strain and rescued the ability to synthesize ubiquinone, suggesting that the eukaryotic CLK-1/Coq7p family could function as bacterial UbiF.     

Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes

The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7). We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F. oxysporum, F. moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella. BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide. While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F. oxysporum Tri101) and FmTri101 (F. moniliforme Tri101) were pseudogenes. Nevertheless, F. oxysporum and F. moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene. By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F. oxysporum; on the basis of this sequence, FmTri201 has been cloned from F. moniliforme by PCR techniques. Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101. The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.     

Mulberry moracins: scavengers of UV stress-generated free radicals

Mulberry leaves treated with UV-C were found to accumulate three different phytoalexins, moracin C, moracin N, and chalcomoracin. The increased level of malondialdehyde in UV-treated leaves along with moracins suggested their role as a free-radical scavenger in stressed plants. All the three moracins induced under UV stress were capable of scavenging the superoxide anion generated by the xanthine-xanthine oxidase system. Also, moracins were capable of inhibiting lipid peroxidation, which strongly indicates their role as a scavenger.     

Facilitatory role of NO in neural norepinephrine release in the rat kidney

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.     

Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis

A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases.     

Binding site on human von Willebrand factor of bitiscetin,a snake venom-derived platelet aggregation inducer

Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans, binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to botrocetin. In contrast with botrocetin which binds to the A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be a bitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor botrocetin had an inhibitory effect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWF monoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to alpha4 helix of the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilized bitiscetin was competitively inhibited by a high concentration of botrocetin. A panel of recombinant VWF, in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1 domain, showed that the bitiscetin-binding activity was reduced in mutations at Arg632, Lys660, Glu666, and Lys673 of the A1 domain. Those substituted at Arg629, Arg636, and Lys667, which decreased the botrocetin binding, showed no effect on the bitiscetin binding. These results indicate that bitiscetin binds to a distinct site in the A1 domain of VWF spanning over alpha4a, alpha5 helices and the loop between alpha5 and beta6 but close to the botrocetin- and NMC-4-binding sites. Monoclonal antibodies recognizing the alpha-subunit of bitiscetin specifically inhibited bitiscetin-induced platelet agglutination without affecting the binding between VWF and bitiscetin, suggesting that the alpha-subunit of bitiscetin is located on VWF closer to the GPIb-binding site than the beta-subunit is. Bitiscetin and botrocetin might modulate VWF by binding to the homologous region of the A1 domain to induce a conformational change leading to an increased accessibility to platelet GPIb.     

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.     

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.     

Time of day improves efficacy and reduces adverse reactions of vitamin D3 in 5/6 nephrectomized rat

Time-dependent differences in adverse reactions and efficacy by a repeated administration of 1,25(OH)2 vitamin D3 (vit D, 0.3 microg/Kg/day for 12 weeks) were examined in 5/6 nephrectomized rats under a condition of 12-hour light-dark cycle. The 5/6 nephrectomy increased serum concentrations of phosphate, osteocalcin and PTH, and urinary excretions of phosphate and deoxypyridinoline (DPD) while the maneuver reduced serum Ca concentration and its urinary excretion. Animals with a dosing of the drug at 2 hours after light on (HALO) had more grade of hypercalcemia and hyperphosphatemia than those at 14 HALO. Reduction of serum intact PTH and increase of serum vit D were observed in both groups with a similar extent. Increase of osteocalcin by the drug was greater in 14 HALO trial. Urinary excretion of DPD was not influenced by the treatment. The increase in bone density of femur was greater in 14 HALO than in 2 HALO trials. These results suggest that adverse reactions of vit D were ameliorated and its efficacy was enhanced after the repeated dosing of the drug at 14 HALO. Time-dependent variation in the sensitivity of the drug to osteoblast was involved in the mechanism of these events, while the roles of pharmacokinetic alteration and renal response were small, if any.