Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.     

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITC-PTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.     

Characteristics of Human Luminance Discrimination and Modeling a Neural Network Based on the Response Properties of the Visual Cortex

Reaction time (RT) and error rate that depend on stimulus duration were measured in a luminance-discrimination reaction time task. Two patches of light with different luminance were presented to participants for ‘short’ (150 ms) or ‘long’ (1 s) period on each trial. When the stimulus duration was ‘short’, the participants responded more rapidly with poorer discrimination performance than they did in the longer duration. The results suggested that different sensory responses in the visual cortices were responsible for the dependence of response speed and accuracy on the stimulus duration during the luminance-discrimination reaction time task. It was shown that the simple winner-take-all-type neural network model receiving transient and sustained stimulus information from the primary visual cortex successfully reproduced RT distributions for correct responses and error rates. Moreover, temporal spike sequences obtained from the model network closely resembled to the neural activity in the monkey prefrontal or parietal area during other visual decision tasks such as motion discrimination and oddball detection tasks.     

Serum surfactant protein-A,but not surfactant protein-D or KL-6, can predict preclinical lung damage induced by smoking

Serum surfactant protein (SP)-A offers a useful clinical marker for interstitial lung disease (ILD). However, SP-A is occasionally elevated in non-ILD pulmonary patients. The present study was conducted to investigate factors that affect serum SP-A levels in respiratory medicine. Serum SP-A, serum SP-D, serum Klebs von den Lungen (KL)-6 and pulmonary function tests were evaluated in 929 patients (current smokers, n=255; ex-smokers, n=242; never-smokers, n=432) without ILD or pulmonary alveolar proteinosis. Serum SP-A was significantly higher in current smokers than in never- or ex-smokers (p<0.01 and p<0.05, respectively). Serum SP-A was significantly higher in chronic obstructive pulmonary disease (COPD) and pulmonary thromboembolism than in other diseases (p<0.01). Serum SP-A correlated positively with amount of smoking (p<0.01) and negatively with forced expiratory volume in 1 s/forced vital capacity (p<0.05). Serum SP-D and KL-6 were unaffected by smoking. Smoking should be taken into account when evaluating serum SP-A levels, and different baseline levels of serum SP-A should be established for smokers and non-smokers. Serum SP-A may also represent a useful marker for predicting COPD in the preclinical stage.     

Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.     

Mrc1 marks early-firing origins and coordinates timing and efficiency of initiation in fission yeast

How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein.     

Impact of Platelet Transfusion on Survival of Patients with Intracerebral Hemorrhage after Administration of Anti-Platelet Agents at a Tertiary Emergency Center

This study examined the impact of platelet transfusion (PLT) on the survival of intracerebral hemorrhage (ICH) patients who had been administered anti-platelet agents (APA). This retrospective cohort analysis investigated 432 patients (259 men, 60%) who were newly diagnosed with ICH between January 2006 and June 2011 at the tertiary emergency center of Kitasato University Hospital. Median age on arrival was 67.0 years (range, 40–95 years). ICH was subcortical in 72 patients (16.7%), supratentorial in 233 (53.9%), and infratentorial in 133 (30.8%). PLT was performed in 16 patients (3.7%). Within 90 days after admission to the center, 178 patients (41.2%) had died due to ICH. Before the onset of ICH, 66 patients had been prescribed APA because of atherosclerotic diseases. Multivariate regression analysis indicated APA administration was an independent risk factor for death within 7 days (odds ratio, 5.12; P = 0.006) and within 90 days (hazard ratio, 1.87; P = 0.006) after arrival. Regarding the effect of a PLT in ICH patients with APA, no patient with PLT died. PLT had a survival benefit on patients with ICH, according to our analysis. Further prospective analysis is necessary to confirm the effects of PLT on survival in ICH with APA.     

Glucocorticoid-induced insulin resistance associates with activation of protein kinase C isoforms

We studied glucocorticoid-induced insulin resistance and possible role of protein kinase C (PKC). Pretreatment with dexamethasone, prednisolone and corticosterone for 60 min decreased insulin-induced [3H] 2-deoxyglucose (DOG) uptake in isolated rat adipocytes. Preincubation with Go6976, LY379196 or myristoylated PKC pseudosubstrate, conventional PKC inhibitor, but not cycloheximide or RU38486, recovered dexamethasone-induced insulin resistance. Dexamethasone activated immunoprecipitates with anti-PKC alpha, beta, and zeta antibodies. PKC zeta activity in adipocytes increased to 163%, and 264% from basal level (100%) with dexamethasone and insulin treatment, respectively. Dexamethasone provoked redistribution of both PKC beta and zeta from the cytosol to the membrane. These results indicate that dexamethasone activates both conventional and atypical PKC. However, conventional PKC is more important in glucocorticoid-induced insulin resistance.     

Slow, tight binding to human renin of some nonpeptidic renin inhibitors containing a 4-methoxymethoxypiperidinylamide at the P4 position

A series of nonpeptidic human renin inhibitors with a 4-methoxymethoxypiperidinylamide at the P4 position of the molecule exhibited slow tight binding to the enzyme. Replacement of the methoxymethoxy moiety on the piperidine ring with H, OH, methoxyethyl, propyloxy or n-butyl eliminated the effect. The inhibition was partially reversed by prolonged dialysis at 4°C, arguing against formation of a covalent bond in the tightened complex.