Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.     

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.     

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.     

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.     

Effect of menstrual cycle phase on the ventilatory response to rising body temperature during exercise

To examine the effect of menstrual cycle on the ventilatory sensitivity to rising body temperature, ten healthy women exercised for ~60 min on a cycle ergometer at 50% of peak oxygen uptake during the follicular and luteal phases of their cycle. Esophageal temperature, mean skin temperature, mean body temperature, minute ventilation, and tidal volume were all significantly higher at baseline and during exercise in the luteal phase than the follicular phase. On the other hand, end-tidal partial pressure of carbon dioxide was significantly lower during exercise in the luteal phase than the follicular phase. Plotting ventilatory parameters against esophageal temperature revealed there to be no significant menstrual cycle-related differences in the slopes or intercepts of the regression lines, although minute ventilation and tidal volume did significantly differ during exercise with mild hyperthermia. To evaluate the cutaneous vasodilatory response, relative laser-Doppler flowmetry values were plotted against mean body temperature, which revealed that the mean body temperature threshold for cutaneous vasodilation was significantly higher in the luteal phase than the follicular phase, but there were no significant differences in the sensitivity or peak values. These results suggest that the menstrual cycle phase influences the cutaneous vasodilatory response during exercise and the ventilatory response at rest and during exercise with mild hyperthermia, but it does not influence ventilatory responses during exercise with moderate hyperthermia.     

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451?bp) following EMA treatment to completely suppress the amplification of DNA of up to 7?logs (10(7)?cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25?U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1?colony forming unit (CFU)/2.22?ml within 7.5?h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1?CFU/2.22?ml).     

Population subdivision of the brackish-water crab Deiratonotus cristatus on the Japanese coast

We investigated the genetic structure of populations of the brackish-water crab Deiratonotus cristatus (de Man, 1895) (family Camptandriidae) on the Japanese coast, together with morphological and the ecological variations. Genetic characteristics of the local populations based on mitochondrial DNA COI sequence data have revealed genetic differentiation between many populations, with the haplotype networks forming three geographical clades: a clade occurring on the Pacific coast, one occurring predominantly in northern Hokkaido, Kyushu and the Seto Inland Sea, and a third occurring in the Ryukyu Islands. Male pleopod morphology, carapace length relative to carapace width, and carapace width of adult crabs varied inconsistently among the geographic groups corresponding to the three clades. Life history traits were similar throughout, although differences in the breeding season were apparent between populations on the Pacific coast, and in the Seto Inland Sea and the Ryukyu Islands.