Further studies on the hyperphosphorylated form (p40) of the rabies virus nominal phosphoprotein (P)

We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.     

Multidimensional protein profiling technology and its application to human plasma proteome

In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.     

Accumulation of copper in kidney of spontaneously hypertensive rats, a preliminary study

Mineral levels such as Cu, Fe, Zn, Ca and Mg in organs of spontaneously hypertensive osteogenic disorder Shionogi rat (SHR-od) were compared with those of normotensive osteogenic disorder Shionogi rat (ODS). The effect of vitamin C deficiency was also examined for both strains. Cu levels in kidney of SHR-od increased to 2.2 times and 5.0 times that of ODS at 6-week old and 30-week old, respectively, although the effect of vitamin C deficiency was not observed in Cu levels of five organs examined. The effect of vitamin C deficiency appeared in the change in the other mineral levels almost similarly for both SHR-od and ODS rats at 30-week old; the decrease of Fe in liver, spleen and lung, whereas the increase of Zn in liver, Ca in heart and Mg in spleen, respectively.     

SMXA-5 mouse as a diabetic model susceptible to feeding a high-fat diet

The SMXA-5 strain, a new mouse model for type 2 diabetes, is a recombinant inbred strain derived from non-diabetic SM/J and A/J strains. As dietary fat is a key component in the development of diabetes, we compared the glucose tolerance and diabetes-related traits among the SMXA-5, SM/J, and A/J strains while feeding a high-fat diet for 10 weeks. SMXA-5 fed on a high-fat diet showed an increased serum insulin concentration. Judging from the hyperinsulinemia in SMXA-5, this strain showed insulin resistance, an inability of peripheral tissues to respond to insulin, which was strengthened by feeding with a high-fat diet. When fed on a high-fat diet for 5 weeks, the SMXA-5 mice showed severely impaired glucose tolerance. On the other hand, SM/J showed mildly impaired glucose tolerance, even when fed on a high-fat diet for 10 weeks. These results indicate that SMXA-5 would be available for use as a diabetic model susceptible to a high-fat diet.     

Glucosyldiacylglycerol enhances reciprocal activation of prourokinase and plasminogen

Reciprocal activation of prourokinase (pro-u-PA) and plasminogen is an important mechanism in the initiation and propagation of local fibrinolytic activity. We found that glucosyldiacylglycerol (GDG) enhanced the reciprocal activation by 1.5- to 2-fold at 0.7-16 microM, accompanying increased conversions of both zymogens to active two-chain forms. The reciprocal activation system consists of (i) plasminogen activation by pro-u-PA to form plasmin, (ii) pro-u-PA activation by the resulting plasmin to form two-chain u-PA (tcu-PA), and (iii) plasminogen activation by the resulting tcu-PA. Whereas GDG minimally affected steps (ii) and (iii) in isolated systems, it markedly enhanced step (i) in the absence of the conversion of pro-u-PA to tcu-PA. GDG significantly increased the intrinsic fluorescence of pro-u-PA (6.7%), but not that of tcu-PA or plasminogen. The large change in intrinsic fluorescence suggests that GDG selectively affects pro-u-PA to alter its conformation, and this mechanism may account for enhancement of its intrinsic plasminogen activator activity.     

Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.     

Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.     

Possible involvement of the p57(Kip2) gene in bone metabolism

We previously uncovered that growth stimulation of rat primary osteoblasts by transforming growth factor-beta1 (TGF-beta1) resulted in a dramatic decrease in p57(Kip2), a member of cyclin-dependent kinase (CDK) inhibitors, through the proteasomal degradation pathway (Urano et al., J. Biol. Chem. 274, 12197-12200, 1999). Here we demonstrated that the amount of p57 protein increases markedly, when rat calvarial primary osteoblasts treated with 1,25-dihydroxyvitamin D3 transit from proliferation toward differentiation. Next, we have analyzed the association of four amino acids deletion polymorphism of p57 and bone mineral density (BMD). The p57 genotype was determined in 154 postmenopausal Japanese women. When we separated the subjects into two groups, one having one or two copies of deletion polymorphism and the other without the deletion, the former subjects had higher BMD (Z score of total body, 0.67 +/- 0.93 vs 0. 23 +/- 0.90, mean +/- standard deviation; P = 0.021). Taken together, these findings suggest that the p57 regulated in the osteoblast proliferation and differentiation may play a role in determination of bone mineral density and pathogenesis of osteoporosis.