Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts

The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.     

Effects of Altered Expression and Localization of Cyclophilin A on Differentiation of p19 Embryonic Carcinoma Cells

1. The retinoblastoma susceptibility gene product, p105Rb (RB), is an important regulator in the control of cell proliferation, differentiation, and apoptosis. Several cellular factors that complex with RB and exert their cellular regulatory functions have been identified, such as the RB:cyclophilin A (CypA) complex. 2. CypA is a cytoplasmic immunophilin and known for its involvement in T-cell differentiation and proliferation. Although CypA has a pivotal role in the immune response, its function in other signaling pathways is largely unknown. 3. In this study, we used a model of neuronal differentiation to demonstrate that the nuclear translocation of CypA, the appearance of hypophosphorylated RB and the enhancement of RB: CypA complex formation correlates with retinoic acid induced neuronal differentiation. 4. Inhibition of CypA expression results in repression of both the hypophosphorylated RB and the neuron-specific differentiation marker, class III beta tubulin. 5. The evidence of enriched CypA and colocalization of RB with CypA in the nucleus of primary adult sensory neurons substantiated the important event of RB-mediated neuronal differentiation of p19 EC cells.     

Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells

We have identified and characterized two mouse cDNAs in a mouse antigen-stimulated bone marrow-derived mast cell cDNA library, both of which encode type I transmembrane proteins. The genes were closely mapped in the distal region of mouse chromosome 11 and expressed not only in mast cells but also widely in leukocytes. The extracellular domains of their encoded proteins contain a single variable immunoglobulin (Ig) motif sharing about 90% identity with amino acids, showing that they comprise a pair of molecules and belong to the Ig superfamily. We named these molecules leukocyte mono-Ig-like receptor1 and 2 (LMIR1 and 2). The intracellular domain of LMIR1 contains several immunoreceptor tyrosine-based inhibition motifs (ITIMs). When cross-linked, the intracellular domain was tyrosine phosphorylated and capable of recruiting tyrosine phosphatases, SHP-1 and SHP-2 and inositol polyphosphate 5-phosphatase, SHIP. LMIR2, on the other hand, contains a short cytoplasmic tail and a characteristic transmembrane domain carrying two positively charged amino acids associated with three kinds of immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecules, DAP10, DAP12, and FcRgamma. These findings suggest that a new pair of ITIM/ITAM-bearing receptors, LMIR1 and 2, regulate mast cell-mediated inflammatory responses through yet to be defined ligand(s).     

Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion

Short interfering RNAs (siRNAs) are powerful sequence-specific reagents that suppress gene expression in mammalian cells. We report for the first time that gene silencing of endothelial E-selectin by siRNAs leads to successful inhibition of leukocyte-endothelial interaction under flow. siRNAs designed to target human E-selectin were tranfected into human umbilical vein endothelial cells (HUVEC). Western blotting analysis revealed that transfection of these siRNAs, but not the scrambled control siRNA (100nM each), attenuated E-selectin expression in HUVEC activated with TNF-alpha (10ng/ml, 4h) without affecting expression of ICAM-1. Moreover, a leukocyte adhesion assay under flow (shear stress=1.0dyne/cm(2)) demonstrated that HUVEC transfected with a siRNA against E-selectin (siE-01) supported significantly less HL60 adhesion as compared to those transfected with the control siRNA (scE-01) after activation (p<0.03). This technique provides a powerful strategy to dissect a specific function of a given molecule in leukocyte-endothelial interaction.     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

Seroepidemiology of hepatitis C virus infection in Japan and HCV infection in haemodialysis patients

Abstract: Since January 1990, Japanese Red Cross Blood Centres have introduced hepatitis C virus screening with a first-generation ELISA. From April to December 1992, approximately 0.98% among 10905 489 blood donations screened by a second-generation assay were anti-HCV-positive in all Japan. Seropositivity of anti-HCV increased with the age and serum transminase value in both sexes. In blood donors having a history of transfusion, the anti-HCV reactive rate was 7.4%. The results of the study made by the Japanese Red Cross Non-A, Non-B Hepatitis Research Group show the effectiveness of implementation of HCV screening to prevent posttransfusion hepatitis. Consecutive haemodialysis patients with chronic renal failure are at risk for inflection by a variety of blood-borne agents transmitted within dialysis units. Because of their immunocompromised state, they frequently also have an unusual susceptibility to a variety of nosocomial infections, such as HBV, and HTLV-I. We tested the prevalence of anti-HCV in 1423 (848 males and 575 females) haemodialiysis patients from 18 hospitals in Kumamoto Prefecture, Japan using the Orhto first generation anti- HCV screening assay. There were 316 patients (22.2%) positive for HCV antibodies. The second-generation test was positive in most haemodialysis patients who were eractive to the firs-generation assay. The prevalence of HCV infection increased with the duration of haemodialysis, yet there was a high frequency of HCV seropositivity even wihtout blood transfusion. Acquisition of HCV in dialysis patients could be explained by HCV seropositivity even without blood (all haemodialysis are done with disposable kits, and needles), by secondary HCV infection after the immunodeficiency of haemodialysis, or by HCV infection of the kidney or glomerular deposition of immune HCV/anti-HCV complexes leading to chronic renal failure (as with HBV infection of the liver and kidney).     

Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

The activation of P2X7 receptor induces cathepsin D-dependent production of a 20-kDa form of IL-1β under acidic extracellular pH in LPS-primed microglial cells

The potent pro-inflammatory cytokine, interleukin-1β (IL-1β), is synthesized as an inactive 33-kDa precursor (pro-IL-1β) and is processed by caspase 1 into the bioactive 17-kDa mature form. The P2X7 receptor, an ATP-gated cation channel, plays an essential role in caspase 1 activation, production and release of mature bioactive 17-kDa form. We recently reported ATP induces the release of an unconventional 20-kDa form of IL-1β (p20-IL-1β) from lipopolysaccharide-primed microglial cells. Emerging evidence suggests physiological relevance for p20-IL-1β; however, the underlying mechanisms for its production and release remain unknown. Here, we investigated the pathways involved in the ATP-induced production of p20-IL-1β using lipopolysaccharide-primed mouse microglial cells. The activation of P2X7 receptor by ATP triggered p20-IL-1β production under acidic extracellular conditions. ATP-induced p20-IL-1β production was blocked by pepstatin A, a potent inhibitor of the lysosomal protease, cathepsin D. The removal of extracellular Ca(2+) inhibited the p20-IL-1β production as well as ATP-induced cathepsin D release via lysosome exocytosis. The acidic extracellular pH also facilitated the dilatation of membrane pore after ATP stimulation. Since facilitation of pore dilatation results in cytolysis accompanied with cytoplasmic pro-IL-1β leakage, our data suggest the leaked pro-IL-1β is processed into p20-IL-1β by cathepsin D released after ATP stimulation under acidic extracellular conditions.     

The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3

We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K_i value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.