HslO ameliorates arrested ΔrecA polA cell growth and reduces DNA damage and oxidative stress responses.

Chromosome damage combined with defective recombinase activity has been widely considered to render cells inviable, owing to deficient double-strand break repair. However, temperature-sensitive recAts polA cells grow well upon induction of DNA damage and supplementation with catalase at restrictive temperatures. These treatments reduce intracellular reactive oxygen species (ROS) levels, which suggests that recAts polA cells are susceptible to ROS, but not chronic chromosome damage. Therefore, we investigated whether polA cells can tolerate a complete lack of recombinase function. We introduced a ΔrecA allele in polA cells in the presence or absence of the hslO-encoding redox molecular chaperon Hsp33 expression plasmid. Induction of the hslO gene with IPTG resulted in increased cell viability in ΔrecA polA cells with the hslO expression plasmid. ΔrecA polA cells in the absence of the hslO expression plasmid showed rich medium sensitivity with increasing ROS levels. Adding catalase to the culture medium considerably rescued growth arrest and decreased ROS. These results suggest that hslO expression manages oxidative stress to an acceptable level in cells with oxidative damage and rescues cell growth. Overall, ROS may regulate several processes, from damage response to cell division, via ROS-sensitive cell metabolism.

Genistein downregulates onco-miR-1260b and inhibits Wnt-signalling in renal cancer cells.

BACKGROUND: Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC). METHODS: Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3'UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes. RESULTS: Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3'UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells. CONCLUSION: Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.

Vitamin C administration attenuates overload-induced skeletal muscle hypertrophy in rats.

AIM: This study aimed to investigate the effects of vitamin C administration on skeletal muscle hypertrophy induced by mechanical overload in rats. METHODS: Male Wistar rats were randomly assigned to three groups: (i) sham-operated group (n = 8), (ii) placebo-administered group (n = 8) and (iii) vitamin C-administered group (n = 8). In the placebo-administered and vitamin C-administered groups, the gastrocnemius and soleus muscles of the right hindlimb were surgically removed to overload the plantaris muscle. Vitamin C (500 mg kg(-1)) was orally administered to the vitamin C-administered group once a day for 14 days. RESULTS: Synergist muscle ablation caused significant increases in wet weight and protein concentration of the plantaris muscle in both the placebo-administered (P < 0.01) and vitamin C-administered groups (P < 0.01) compared with the sham-operated group (SHA). However, the magnitude of plantaris muscle hypertrophy (expressed as a percentage of the contralateral plantaris muscle) was significantly smaller (P < 0.01) in the vitamin C-administered group (141%) than in the placebo-administered group (PLA) (152%). Compared with the SHA, only the PLA showed higher expressions of phosphorylated p70s6k and Erk1/2 (positive regulators of muscle protein synthesis) and a lower expression of atrogin-1 (a muscle atrophy marker). Concentrations of vitamin C and oxidative stress markers in the overloaded muscle were similar between the placebo-administered and vitamin C-administered groups. CONCLUSION: Oral vitamin C administration can attenuate overload-induced skeletal muscle hypertrophy, which may have implications for antioxidant supplementation during exercise training.

Grape seed proanthocyanidin lowers brain oxidative stress in adult and middle-aged rats.

There is growing concern over the increasing instances of decline in cognitive abilities with aging in humans. The present study evaluated the benefits of the natural antioxidant, grape seed proanthocyanidin extract (GSPE) in treating the effects of age-related oxidative stress (OS) and accumulation of lipofuscin (LF) on the cognitive ability in rats. Female Wistar rats of 3- and 12-months of age received a daily oral supplement of GSPE until they attained 6- and 15-months of age. During this period, rats were tested for their cognitive ability. At the end of this period, blood glucose and markers of OS were assessed in the hippocampus. GSPE lowered blood glucose, lipid peroxidation, hydrogen peroxide level, and increased protein sulphydryl (P-SH) content in the hippocampus. In addition, GSPE significantly improved cognitive performance in the two age groups. These results demonstrate that the extent of OS-related LF accumulation is reducible by GSPE. They also suggest a critical role for GSPE as a neuroprotectant in the hippocampus and in preventing cognitive loss with aging.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses.

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

Characterization and chromosomal mapping of a novel human gene, ANKHZN.

Ankhzn (ankyrin repeats hooked to a zinc finger motif) was originally isolated by means of the gene trap method, as a novel cytoplasmic protein on mouse embryonic stem cells. The Ankhzn protein is ubiquitously expressed in a spatiotemporal-specific manner and is located on endosomes. In the present study, we have cloned human ANKHZN cDNA by PCR using candidate EST clones exhibiting a high homology to mouse Ankhzn cDNA. The human ANKHZN cDNA encoded a 1166aa protein exhibiting 84.9% identity to the mouse one. The size of the transcript was found to be about 7kb on a Northern blot analysis, and ANKHZN mRNA was found to be ubiquitously expressed in human tissues on RT-PCR analysis. Western blot analysis showed that a 130kDa protein was detected at various levels in human tissues and also present in both membrane and soluble fractions obtained on subcellular fractionation. Human ANKHZN is a single copy gene consisting of predicted 25 exons in the human genome, and has been mapped to human chromosome 17p13 by radiation hybrid panel and fluorescence in-situ hybridization.

Molecular cloning of a novel 130-kDa cytoplasmic protein, Ankhzn, containing Ankyrin repeats hooked to a zinc finger motif.

A novel gene was trapped in mouse embryonic stem cells with a promoterless gene trap vector. Fused transcripts were isolated from the embryos by rapid amplification of cDNA ends, which were used for full-length cDNA cloning. The protein predicted from the cDNA consisting of 7143 nucleotides comprises 1184 amino acids, which was confirmed by in vitro transcription/translation assaying. An antibody against the synthesized peptide reacted with an approximate 130-kDa protein on SDS-PAGE. A search of available databases revealed that this protein is a novel protein composed of 17 ankyrin repeats hooked to a zinc finger motif, which we named Ankhzn. Ankhzn was observed on the endosomal membrane on immunoelectron microscopic analysis. Ankhzn belongs to a new subgroup of double zinc finger proteins which may be involved in vesicle or protein transport. Ankhzn mRNA and its protein were expressed ubiquitously from embryonic day 10.5 to adulthood.

A novel superoxide dismutase gene encoding membrane-bound and extracellular isoforms by alternative splicing in Caenorhabditis elegans.

We have identified a novel Cu/Zn superoxide dismutase gene (termed SOD-4) in Caenorhabditis elegans. Characterization of its complementary DNA revealed that the gene encodes two isoforms by alternative splicing, SOD4-1 and SOD4-2 which differ in their C-terminal exons. Their predicted amino acid sequences include a consensus signal peptide at their N-termini and are homologous to the extracellular-types of Cu/Zn superoxide dismutase in mammals. In addition, SOD4-2 possesses a putative transmembrane domain at the C-terminal region. When transiently expressed in Chinese hamster ovary cells, both types were found in the membranes and SOD4-1 also in the culture fluid. It is, therefore, indicated that SOD4-1 is an extracellular form and SOD4-2 a membrane-bound form, the latter representing a novel type of SOD. In C. elegans, SOD4-2 mRNA was found to be preferentially expressed in eggs.

Cloning, sequencing and mapping of a manganese superoxide dismutase gene of the nematode Caenorhabditis elegans.

We have cloned, sequenced and mapped a gene (sod-2) encoding manganese superoxide dismutase [EC 1.15.1.1] from the nematode Caenorhabditis elegans. The sod-2 was mapped to chromosome I by hybridization with a YAC polytene filter. The protein-coding region spans 1129 base pairs including 4 introns and encodes a protein of 221 amino acids (aa) (M(r) = 24536) of which the first 24 aa are the presumed mitochondorial-targeting signal peptide. The gene sequence of sod-2 was slightly different from an isoform, sod-3.

Pruritic papular eruption of the acquired immunodeficiency syndrome.

We report a case of acquired immunodeficiency syndrome with pruritic papular eruption. The patient, a hemophiliac, presented with generalized pruritic, skin-colored papules and nodules. The chronic lesions were excoriated and hyper-pigmented. The eosinophil count was elevated, but IgE was normal. The lesions and pruritus responded only to ultraviolet B phototherapy. While the mechanism is not known, ultraviolet B phototherapy may provide relief of AIDS-related pruritus.

Pancreatic tissue damage by transcatheter arterial embolization for hepatoma.

We analyzed the serial changes in serum pancreatic enzyme activities by transcatheter arterial embolization (TAE) in 20 hepatoma patients with liver cirrhosis in an attempt to evaluate the incidence of the pancreatic tissue damage by TAE. Serum amylase activities increased in two (10%) cases, elastase 1 levels in six (30%) cases, and trypsin and pancreatic secretory trypsin inhibitor (PSTI) levels in each of five (25%) cases. Consequently, TAE resulted in the elevation of at least more than one serum pancreatic enzyme in eight (40%) of 20 cases, although none had clinical symptoms related to pancreatitis. When the adverse effect on the pancreatic tissue was compared among 6 cases of the superselective TAE and 14 cases of the nonsuperselective TAE, which were performed from the segmental and the nonsegmental hepatic arteries, respectively, the elevation of serum pancreatic enzymes was caused only by nonsuperselective TAE, not by superselective TAE. The volumes of Spongel and Lipiodol used or the injected doses of the anticancer agent mitomycin C were not different between the two groups. These results indicate that TAE for the treatment of hepatoma frequently causes pancreatic tissue damage, and the position of the inserted catheter tip is very important to avoid the pancreatic tissue damage by TAE.

Electrical stimulation of pelvic floor musculature by percutaneous implantable electrodes: a case report.

A forty-year-old man with reflex urinary incontinence due to spinal cord injury was treated with electrical stimulation of the pelvic floor musculature. In this case we employed percutaneous implantable electrodes and an external pulse regulator. After 4 weeks of stimulation incontinence was improved and urodynamically maximum cystometric capacity increased from 220 ml to 350 ml. Our method is easy and not invasive. This technique can be an alternative for the electrical stimulation for urinary incontinence.

Changes in sarcoplasmic metabolite concentrations and pH associated with the catch contraction and relaxation of the anterior byssus retractor muscle of Mytilus edulis measured by phosphorus-31 nuclear magnetic resonance.

The sarcoplasmic concentrations of phosphorus metabolites and pH (pHin) were measured in the anterior byssus retractor muscle (ABRM) of Mytilus edulis by 31P nuclear magnetic resonance spectroscopy. During an active contraction induced by 10(-3) acetylcholine, the concentration of arginine phosphate ([Arg-P]in) decreased from the resting value of 7.47 +/- 0.26 (mean +/- SE, n = 8) to 6.67 +/- 0.29 (n = 6) mumol g-1, and that of inorganic phosphate (Pi) consistently increased from 0.84 +/- 0.06 (n = 7) to 1.61 +/- 0.12 (n = 5) mumol g-1. In the 'catch' state following the active contraction, these concentrations were close to their resting levels, indicating that the catch is an inactive state. 5-hydroxytryptamine caused a rapid relaxation of the catch, which was associated with a slight decrease in [Arg-P]in and an increase in pHin by ca 0.2 units. The sarcoplasmic concentration of ATP (mean, 1.6 mumol g-1) did not change throughout the contraction-relaxation cycle.

Therapeutic effect of aspoxicillin on experimental pneumonia with Klebsiella pneumoniae in mice.

The therapeutic effects of aspoxicillin (ASPC) on an experimental pneumonia in mice were compared with those of piperacillin (PIPC) and mezlocillin (MZPC) under various administration schedules. The pneumonia was induced with K. pneumoniae B-54 by the aerosol method. Fifty mg/kg of each penicillin was subcutaneously injected into mice starting from 12 h after infection. At 3- and 6-h interval regimens, ASPC caused the infected mice to survive longer than the other penicillins. The decrease of viable bacterial counts in the lung after a single or repeated injection of ASPC occurred more rapidly than with the other drugs. The concentration of ASPC in the lung after a single injection was higher than that of the other drugs and the concentration was maintained above the MIC for about 2 h. The therapeutic effects of these penicillins on this model reflected well their concentrations in the lung. Among these penicillins, ASPC gave the highest maximum level and persisted longest in the lung, so is shown to have a therapeutic effect superior to PIPC and MZPC on this model of pneumonia. The findings obtained in this experimental pneumonia model were concluded to correlate well with the good clinical efficacy of ASPC compared to PIPC.

Effect of intrahepatic arterial infusion of 131I-labelled lipiodol on hepatocellular carcinoma of rat.

Intrahepatic arterial infusion of 131I-labelled lipiodol was performed to study the intrahepatic distribution of lipiodol and to determine the radiation effect on 3'-Methyl-4-Dimethylaminobenzene (DAB) induced hepatocellular carcinoma (HCC) in rats. From the findings of the softex films and scintigrams of the liver, according to the time course, lipiodol was found to accumulate in the tumour tissues parallel with the degree of perfusion, and it remained in the tumour tissues for an extended period probably due to the delay of the degradation of lipiodol compared to that in non tumour tissues. It was noticed that the lipiodol, accumulated and deposited selectively in the tumour tissues, existed mostly in the extracellular space but not in the intracellular space. In histological studies of the resected specimens of the tumours, complete necrosis of hepatocellular carcinoma was recognized 2 to 8 weeks after the infusion of 131I-labelled lipiodol, while there was none in the control group where cold lipiodol was employed. These results suggest that the most effect on hepatocellular carcinoma in this study is caused by the radiation effect of 131I-labelled lipiodol.