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.     

Developmental switch from GABA to glycine release in single central synaptic terminals

Early in postnatal development, inhibitory inputs to rat lateral superior olive (LSO) neurons change from releasing predominantly GABA to releasing predominantly glycine into the synapse. Here we show that spontaneous miniature inhibitory postsynaptic currents (mIPSCs) also change from GABAergic to glycinergic over the first two postnatal weeks. Many 'mixed' mIPSCs, resulting from co-release of glycine and GABA from the same vesicles, are seen during this transition. Immunohistochemistry showed that a large number of terminals contained both GABA and glycine at postnatal day 8 (P8). By P14, both the content of GABA in these mixed terminals and the contribution of GABA to the mixed mIPSCs had decreased. The content of glycine in terminals increased over the same period. Our results indicate that switching from GABAergic to glycinergic inputs to the LSO may occur at the level of a single presynaptic terminal. This demonstrates a new form of developmental plasticity at the level of a single central synapse.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1-/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1-/- mice, and IFN-gamma that was overproduced in WSX-1-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-alpha, were produced by liver mononuclear cells in WSX-1-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production.     

Bilateral receptive field neurons in the hindlimb region of the postcentral somatosensory cortex in awake macaque monkeys

Single-neuron activities were recorded in the hindlimb region of the primary somatosensory cortex and part of area 5 in awake Japanese monkeys. A total of 1050 units were isolated from five hemispheres of four animals. Receptive fields (RFs) and submodalities were identified for 90% of isolated neurons in areas 3a and 3b. The percentage decreased as the recoding site moved to the more caudal areas. Deep or skin submodality neurons were dominant in area 3a or area 3b, respectively. Deep submodality neurons increased in more caudal areas and were the majority in areas 2 and 5. These observations were consistent with those in the hand and/or digit or arm and/or trunk region. The identified neurons were classified by their RF positions into four types: the foot, leg, foot and leg, or hindlimb and other body parts type. Among 831 identified neurons, 33 neurons had bilateral RFs, 14 had ipsilateral RFs, and the rest (N=784) had contralateral RFs. The relative incidence of neurons with bilateral or ipsilateral RFs among identified neurons was less than 1% in areas 3a, 3b, and 1, and 16% or 25% in areas 2 or 5, respectively. Within areas 2 and 5, the percentage of neurons with bilateral or ipsilateral RFs was significantly smaller in the foot type (5%) than in other RF types (24-57%). RFs of the foot type were on the sole or single toe but never on multiple toes. These observations contrasted with the previous findings that neurons with bilateral RFs were more frequently seen in the hand and/or digit region and that RFs on multiple digit tips were dominant there. The present study thus demonstrated that neurons with bilateral RFs do exist in the hindlimb region. Similarly to the forelimb region, they were found mostly in areas 2 and 5, the caudalmost areas of the postcentral gyrus and hierarchically higher stages in information processing. The relative paucity of neurons with bilateral RFs on the foot, especially those with RFs on multiple toes, may reflect functional differences between the foot and the hand.