Molecular cloning of acetyl coenzyme A: deacetylcephalosporin C o-acetyltransferase cDNA from Acremonium chrysogenum: sequence and expression of catalytic activity in yeast.

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C, the conversion of deacetylcephalosporin C to cephalosporin C. A cDNA encoding DCPC-ATF has been isolated from a cDNA library of a cephalosporin C producing fungus Acremonium chrysogenum using oligonucleotide probes based on N-terminal amino acid sequences of the enzyme. The cDNA contains a single large open reading frame for a putative precursor consisting of 12 amino acid(AA) leader peptide of unknown function, 274 AA large subunit and 126 AA small subunit at the carboxyl end. The cDNA was expressed in yeast exhibiting a functional DCPC-ATF activity. It was also indicated that the leader peptide was not essential for expression of the enzyme activity. The primary structure of DCPC-ATF shows significant homology with those of acetyl CoA: homoserine o-acetyltransferase in Saccharomyces cerevisiae and Ascobolas immersus.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Relationship between general or specific immunoreactivity and prognosis in postoperative patients with hepatocellular carcinoma

Summary The levels of a variety of immunological parameters were examined in 203 preoperative patients with hepatocellular carcinoma (HCC) at various stages (I–IV). The changes in the peripheral blood lymphocyte (PBL) count, the serum level of immunosuppressive acidic protein and the degree of the skin reaction to purified protein derivative were associated significantly with the stage of HCC progression. However, the percentages of lymphocyte subsets, mitogenic responsiveness of PBL and serum immunoglobulin concentration remained at the levels of stage I. Further study demonstrated that in patients undergoing hepatic artery ligation, there were statistically significant correlations between the PBL count, immunosuppressive acidic protein concentration and intensity of the skin reaction to purified protein derivative, assayed 1 month after surgery, and the prognosis. HCC-specific immunity was examined in 34 patients treated by hepatic resection or hepatic artery ligation using in vitro responses of PBL to HCC extracts (ATS test). This test was performed using culture medium containing added arginine. None of the PBL from the patients showed a positive response to allogeneic HCC extracts, but the PBL from 12 patients (9 hepatic resections, 3 hepatic artery ligations) were stimulated significantly (SI 2.5) with autologous HCC extracts. In 7 of 9 hepatic resection patients who were positive in the ATS test, tumor recurrence was identified. Statistical analysis indicated that the ATS test result was significantly correlated with tumor recurrence in hepatic resection patients. Autologous-PBL-stimulating activities were isolated in a fraction at pH 8.3 and in fractions at pH 6.7–7.0 by chromatofocusing of the crude extract. Although identification of the HCC-specific antigen remains to be done, use of the above fractions may simplify the ATS test procedure and improve its sensitivity.     

Nutrient-dependent requirement for SOD1 in lifespan extension by protein restriction in Drosophila melanogaster

Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target of rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly, rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP, and low-calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.     

Longitudinal Study of the Decline in Renal Function in Healthy Subjects

Background

Chronic kidney disease is an important concern in preventive medicine, but the rate of decline in renal function in healthy population is not well defined. The purpose of this study was to determine reference values for the estimated glomerular filtration rate (eGFR) and rate of decline of eGFR in healthy subjects and to evaluate factors associated with this decline using a large cohort in Japan.

Methods

Retrospective cross-sectional and longitudinal studies were performed with healthy subjects aged ≥18 years old who received a medical checkup. Reference values for eGFR were obtained using a nonparametric method and those for decline of eGFR were calculated by mixed model analysis. Relationships of eGFR decline rate with baseline variables were examined using a linear least-squares method.

Results

In the cross-sectional study, reference values for eGFR were obtained by gender and age in 72,521 healthy subjects. The mean (±SD) eGFR was 83.7±14.7ml/min/1.73m². In the longitudinal study, reference values for eGFR decline rate were obtained by gender, age, and renal stage in 45,586 healthy subjects. In the same renal stage, there was little difference in the rate of decline regardless of age. The decline in eGFR depended on the renal stage and was strongly related to baseline eGFR, with a faster decline with a higher baseline eGFR and a slower decline with a lower baseline eGFR. The mean (±SD) eGFR decline rate was ‒1.07±0.42ml/min/1.73m²/year (‒1.29±0.41%/year) in subjects with a mean eGFR of 81.5±11.6ml/min/1.73m².

Conclusions

The present study clarified for the first time the reference values for the rate of eGFR decline stratified by gender, age, and renal stage in healthy subjects. The rate of eGFR decline depended mainly on baseline eGFR, but not on age, with a slower decline with a lower baseline eGFR.     

Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Structural basis for sorting mechanism of p62 in selective autophagy

Impairment of autophagic degradation of the ubiquitin- and LC3-binding protein "p62" leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser334-Ser344) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56 angstroms resolution revealed interaction of Trp340 and Leu343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.     

Zinc sensing for cellular application

Numerous tools for Zn2+ sensing in living cells have become available in the past three years. Among them, fluorescence imaging using fluorescent sensor molecules has been the most popular approach. Some of these sensor molecules can be used to visualize Zn2+ in living cells. Some of the biological functions of Zn2+ have been clarified using these sensor molecules, especially in neuronal cells, which contain a high concentration of free Zn2+.     

Accelerated biodegradation of poly(vinyl alcohol) by glycosidations of the hydroxyl groups or addition of sugars

Biodegradabilities of N-acetyl-d-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 degrees C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP = 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, (1)H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAc-substituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability.     

Change in sleep state of the elderly before and after cataract surgery

It seems likely that the influences of light upon circadian rhythms will decrease with aging, particularly those rhythms that are more influenced by light with a higher color temperature and richer in short wavelengths. More specifically, cataract patients' optical systems transmit light poorly, especially the shorter wavelengths that affect the circadian system more. The present study investigated melatonin secretion profiles and sleep patterns before and after cataract surgery. Fifteen subjects were studied for 3 consecutive weekdays before, and one month after, their cataract surgery. UV-cutting intra-ocular lenses were used for patients after surgery. No statistically significant differences between before and after surgery were observed in the amount of light received and the amount of activity. This means that there were no significant changes in their lifestyle during the experimental period. Considering the group as a whole, no significant differences were present in melatonin secretion, sleep parameters, or sleepiness before and after the surgery. However, individual subjects responded differently. The subjects showed a negative correlation between the wake-up (p=0.067) or retiring times (p=0.017) and sleep efficiency after surgery. The amount of light received during the nighttime influenced sleep more significantly than during the daytime.