Effect of ATP on the Release of hsp 70 and hsp 40 from the Nucleus in Heat-Shocked HeLa Cells

We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunoflourescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.     

Salt intake and mental distress among rural community-dwelling Japanese men

Background

Activated mineralocorticoid receptors influence the association between daily salt intake and blood pressure. A relatively low mineralocorticoid receptor function is reported to be a risk for mental distress such as depression. Since mental distress is also a known risk for hypertension and cardiovascular disease, understanding of the association between estimated daily salt intake and mental distress contributing to hypertension is important for risk estimation for cardiovascular disease. However, no single study has reported this association.

Methods

We conducted a cross-sectional study of 1014 Japanese men undergoing general health check-ups. Mental distress was diagnosed as a Kessler 6 scale score ≥5. We also classified mental distress by levels of hypertension. Estimated daily salt intake was calculated from a causal urine specimen.

Results

Independent from classical cardiovascular risk factors and thyroid disease, we found a significant inverse association between estimated daily salt intake and mental distress. When we analyzed for mental distress and hypertension, we also found a significant association. With the reference group being the lowest tertiles of estimated daily salt intake, the multivariable odds ratios (ORs) of mental distress and mental distress with hypertension for the highest tertiles were 0.50 (0.29–0.88) and 0.46 (0.22–0.96).

Conclusions

Lower estimated daily salt intake is a significant risk of mental distress for rural community-dwelling Japanese men. Since depression is reported to be associated with cardiovascular disease, risk estimation for the lower intake of salt on mental distress, especially for mental distress with hypertension, may become an important tool to prevent cardiovascular disease.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

Potent anti-R5 human immunodeficiency virus type 1 effects of a CCR5 antagonist, AK602/ONO4128/GW873140, in a novel human peripheral blood mononuclear cell nonobese diabetic-SCID, interleukin-2 receptor gamma-chain-knocked-out AIDS mouse model

We established human peripheral blood mononuclear cell (PBMC)-transplanted R5 human immunodeficiency virus type 1 isolate JR-FL (HIV-1(JR-FL))-infected, nonobese diabetic-SCID, interleukin 2 receptor gamma-chain-knocked-out (NOG) mice, in which massive and systemic HIV-1 infection occurred. The susceptibility of the implanted PBMC to the infectivity and cytopathic effect of R5 HIV-1 appeared to stem from hyperactivation of the PBMC, which rapidly proliferated and expressed high levels of CCR5. When a novel spirodiketopiperazine-containing CCR5 inhibitor, AK602/ONO4128/GW873140 (molecular weight, 614), was administered to the NOG mice 1 day after R5 HIV-1 inoculation, the replication and cytopathic effects of R5 HIV-1 were significantly suppressed. In saline-treated mice (n = 7), the mean human CD4(+)/CD8(+) cell ratio was 0.1 on day 16 after inoculation, while levels in mice (n = 8) administered AK602 had a mean value of 0.92, comparable to levels in uninfected mice (n = 7). The mean number of HIV-RNA copies in plasma in saline-treated mice were approximately 10(6)/ml on day 16, while levels in AK602-treated mice were 1.27 x 10(3)/ml (P = 0.001). AK602 also significantly suppressed the number of proviral DNA copies and serum p24 levels (P = 0.001). These data suggest that the present NOG mouse system should serve as a small-animal AIDS model and warrant that AK602 be further developed as a potential therapeutic for HIV-1 infection.