Prospective clinical evaluation of the serologic tuberculous glycolipid test in combination with the nucleic acid amplification test

We have conducted a prospective controlled multicenter study to evaluate differences in the levels of clinical utility of the tuberculous glycolipid (TBGL) serodiagnostic test and the nucleic acid amplification test in patients with smear-negative active pulmonary tuberculosis (TB). The TBGL test and the PCR test were individually not so useful for the rapid diagnosis of smear-negative active pulmonary TB. However, clinical utility was considerably improved by using the TBGL test and the PCR test in combination, especially in patients with smear-negative and culture-negative active pulmonary TB and in patients with minimally advanced lesions.     

Pathogenic capacity of proteases from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin.

The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vascular permeability, which was followed by edema formation. The permeability-enhancing activity of the proteases increased in parallel with the concentration of the enzymes. When tested in vitro for its effect on these bacterial proteases, chicken egg white ovomacroglobulin (ovoM) inhibited the enzymatic activity of all the proteases after a short incubation period at an enzyme/inhibitor ratio (molar) of 1:1 to 1:4 or at a lower concentration after a longer incubation period. Such treatment of the proteases with chicken egg white ovoM before injection intrastromally into the eyes or intradermally into the clipped flanks of guinea pigs protected the cornea from destruction or completely prevented the permeability reaction and edema formation. No inhibitory effects of plasma protease inhibitors against these bacterial proteases were noted. Since the proteases are critical in the pathogenic processes caused by the bacteria, these results suggest a beneficial effect of ovoM against bacterial infections.     

Study on effects of life review activities on the quality of life of the elderly: a randomized controlled trial

BACKGROUND: The objective of this study was to evaluate the mid-term efficacy of life review activities on the quality of life (QOL) of the elderly by conducting a randomized controlled trial, and to identify the factors that should be taken into consideration when conducting life review activities. METHODS: Written consent was obtained from 80 of the 97 eligible elderly persons. After randomly assigning them to two groups, an intervention group and a control group, group life review activities were conducted in the intervention group and discussion activities about health were conducted in the control group. In both the intervention group and the control group, life satisfaction, self-esteem, depression, and hopelessness were evaluated using self-rating scales at three points: at baseline, immediately after completion of the 8 weeks of sessions, and 3 months after completion of the intervention. RESULTS: Repeated measures analysis of covariance showed significant differences between the two groups in the changes in scores for depression (p = 0.04) and hopelessness (p = 0.04). Regarding the factors that were associated with depression and hopelessness, 3 months after completion of the intervention, depression and hopelessness of a more severe nature at baseline and having greater unresolved conflicts in the past were extracted by multiple regression analysis. CONCLUSIONS: The results suggested that group life review activities have a role in assisting the developmental stage of old age and supporting mental health, and have mid- to long-term effectiveness in maintaining and improving the QOL of the elderly.     

HeLa cell invasiveness and O antigen of Shigella flexneri as separate and prerequisite attributes of virulence to evoke keratoconjunctivitis in guinea pigs.

Many rough mutants selected from isogenic smooth virulent and avirulent strains of Shigella flexneri were examined for virulence, using tissue culture infection and Sereny tests. Many of the rough mutants isolated from a virulent smooth strain were capable of penetrating tissue culture cells but incapable of producing a positive Sereny test. In contrast, we could not obtain from smooth avirulent strains any rough mutants capable of penetrating HeLa cells. Chemical analysis of lipopolysaccharide of some representative rough strains showed several patterns of sugar composition with a range of from Ra to Re chemotypes. There was no correlation between HeLa cell invasiveness and chemotypes of lipopolysaccharides, thus indicating little significance of oligosaccharides of the rough core as well as O antigens in the ability of S. flexneri to penetrate HeLa cells. When these invasive rough strains were given O antigen genes from a smooth avirulent Shigella Hfr strain, most of the transconjugants that expressed O antigens regained the ability to evoke keratoconjunctivitis in guinea pigs. We also examined the chromosomal loci of HeLa cell invasion by transferring carbohydrate fermentation genes of Escherichia coli K-12 Hfr and found two chromosomal loci, the rha and lac-gal regions, which control the ability to penetrate HeLa cells. These results suggested that O antigens and ability to penetrate tissue culture cells are independent and prerequisite attributes of virulence in Shigella flexneri to evoke keratoconjunctivitis in guinea pigs.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation

Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)72 repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. Poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(Q)(72) aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.     

ARK5 expression in colorectal cancer and its implications for tumor progression

A novel member of the human AMPK family, ARK5, was recently discovered to be a key molecule in mediating cancer cell migration activity in human pancreas cancer cell line PANC-1, and its activation was found to be induced by Akt-dependent phosphorylation at Ser 600. DNA array analysis with 241 paired cDNAs from 13 different types of tumors and corresponding normal tissues derived from cancer patients revealed ARK5 overexpression in the samples of colorectal cancer. ARK5 expression was measured and an in vitro invasion assay was performed in six human colorectal cancer cell lines, WiDr, HCT-15, DLD-1, SW620, LoVo, and SW480, and since high invasion activity was concordant with higher ARK5 expression, ARK5 expression was examined in relation to tumor progression and metastatic activity in clinical samples. In 56 clinical samples of primary colorectal cancers and their liver metastases, higher ARK5 expression was observed in the samples from more advanced cases, and much higher expression was observed in the liver metastases. In situ hybridization analysis showed ARK5 overexpression in tumor cells. Based on these findings, we propose that ARK5 overexpression is involved in tumor progression of colon cancer clinically.