Recent advances in p97/VCP/Cdc48 cellular functions

p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.     

Repetitive transcranial magnetic stimulation induces kf-1 expression in the rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive approach used for stimulating the brain, and has proven effective in the treatment of depression, however the mechanism of its antidepressant action is unknown. Recently, we have reported the induction of kf-1 in rat frontal cortex and hippocampus after chronic antidepressant treatment and repeated electroconvulsive treatment (ECT). In this study, we demonstrated the induction of kf-1 after rTMS in the rat frontal cortex and hippocampus, but not in hypothalamus. Our data suggest that kf-1 may be a common functional molecule that is increased after antidepressant treatment, ECT and rTMS. In conclusion, it is proposed that induction of kf-1 may be associated with the treatment induced adaptive neural plasticity in the brain, which is a long-term target for their antidepressant action.     

Expression and characterization of Saccharomyces cerevisiae Cne1p, a calnexin homologue

The calnexin homologue (Cne1p) of Saccharomyces cerevisiae was expressed in Escherichia coli to evaluate its chaperone function. The chaperone function was examined as to the effects on the suppression of thermal denaturation and the enhancement of refolding, using citrate synthase (CS) as a nonspecific chaperone substrate. Cne1p effectively suppressed the thermal denaturation of CS and enhanced the refolding of thermally or chemically denatured CS in a concentration-dependent manner. In addition, the chaperone function of Cne1p was greatly affected in the presence of monoglucosylated oligosaccharides (G1M9) that specifically bind to the lectin site. These results indicated that Cne1p functions as a molecular chaperone in Saccharomyces cerevisiae.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Photophosphorylation and oxidative phosphorylation in intact cells and chromatophores of an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Light-induced ATP synthesis was studied in intact cells and chromatophores of Erythrobacter sp. strain OCh114. ATP synthesis was measured by both the pH method and the luciferin-luciferase luminescence method. The rate of ATP synthesis was moderate (a typical value of 0.65 mol of ATP per mol of bacteriochlorophyll per min), and synthesis was inhibited by antimycin A. ATP was synthesized under illumination only under aerobic conditions and not under anaerobic conditions. This characteristic was similar to that of other light-induced energy transduction processes in this bacterial species, such as oxidation of reaction center, oxidation of cytochrome c551, and translocation of H+, which were not observed under anaerobic conditions. This phenomenon was reconciled with the fact that the Erythrobacter sp. could not grow anaerobically even in the light. The characteristics of oxidative phosphorylation and ATP hydrolysis were also investigated. The respiratory ratio of chromatophores was 2.3. Typical rates of oxidative phosphorylation by NADH and by succinate were 2.9 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.22) and 1.1 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.19), respectively. A typical rate of ATP hydrolysis was 0.25 mol of ATP per mol of bacteriochlorophyll per min in chromatophores. ATPase and adenylate kinase are also involved in the metabolism of adenine nucleotides in this bacterium.     

Effect of Behavior Modification on Outcome in Early- to Moderate-Stage Chronic Kidney Disease: A Cluster-Randomized Trial

Objectives

Owing to recent changes in our understanding of the underlying cause of chronic kidney disease (CKD), the importance of lifestyle modification for preventing the progression of kidney dysfunction and complications has become obvious. In addition, effective cooperation between general physicians (GPs) and nephrologists is essential to ensure a better care system for CKD treatment. In this cluster-randomized study, we studied the effect of behavior modification on the outcome of early- to moderate-stage CKD.

Design

Stratified open cluster-randomized trial.

Setting

A total of 489 GPs belonging to 49 local medical associations (clusters) in Japan.

Participants

A total of 2,379 patients (1,195 in group A (standard intervention) and 1,184 in group B (advanced intervention)) aged between 40 and 74 years, who had CKD and were under consultation with GPs.

Intervention

All patients were managed in accordance with the current CKD guidelines. The group B clusters received three additional interventions: patients received both educational intervention for lifestyle modification and a CKD status letter, attempting to prevent their withdrawal from treatment, and the group B GPs received data sheets to facilitate reducing the gap between target and practice.

Main outcome measure

The primary outcome measures were 1) the non-adherence rate of accepting continuous medical follow-up of the patients, 2) the collaboration rate between GPs and nephrologists, and 3) the progression of CKD.

Results

The rate of discontinuous clinical visits was significantly lower in group B (16.2% in group A vs. 11.5% in group B, p = 0.01). Significantly higher referral and co-treatment rates were observed in group B (p<0.01). The average eGFR deterioration rate tended to be lower in group B (group A: 2.6±5.8 ml/min/1.73 m²/year, group B: 2.4±5.1 ml/min/1.73 m²/year, p = 0.07). A significant difference in eGFR deterioration rate was observed in subjects with Stage 3 CKD (group A: 2.4±5.9 ml/min/1.73 m²/year, group B: 1.9±4.4 ml/min/1.73 m²/year, p = 0.03).

Conclusion

Our care system achieved behavior modification of CKD patients, namely, significantly lower discontinuous clinical visits, and behavior modification of both GPs and nephrologists, namely significantly higher referral and co-treatment rates, resulting in the retardation of CKD progression, especially in patients with proteinuric Stage 3 CKD.

Trial registration

The University Hospital Medical Information Network clinical trials registry UMIN000001159