Purification of a 29-kDa hemocyte proteinase of Sarcophaga peregrina.

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of cathepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.     

Induction of TNF-like factor by murine macrophage-like cell line J774.1 on treatment with Sarcophaga lectin

On stimulation with Sarcophaga lectin, the mouse macrophage-like cell line J774.1 secreted a factor like the tumor necrosis factor (TNF) into the culture medium. This factor was a protein with a molecular weight of 40000-45000, and was cytotoxic to L-929 cells, but not to normal embryonic fibroblasts. This factor was effective on both the ascites form and solid form of sarcoma 180 transplanted into ICR mice.     

Statin reverses reduction of adiponectin receptor expression in infarcted heart and in TNF-alpha-treated cardiomyocytes in association with improved glucose uptake

Statin treatment improves insulin resistance in skeletal muscle. Thus this study assessed whether statin may affect the myocardial expression levels of AdipoR1 and AdipoR2, receptors of adiponectin that enhance insulin sensitivity, and whether statin may improve insulin resistance in cardiomyocytes. Myocardial infarction (MI) was created by the ligation of the left coronary artery in male mice. Expression levels of mRNA and protein levels of AdipoR1 but not of AdipoR2 were significantly decreased in the remote area as well as in the healed infarcted area in the left ventricles 4 wk after MI. Oral administration of pravastatin (50 mg.kg(-1).day(-1) for 4 wk after MI) reversed the decrease in myocardial expression levels of AdipoR1 independently of changes in serum lipid profiles and insulin levels. With the use of cultured cardiomyocytes, incubation with tumor necrosis factor (TNF)-alpha, a mediator of postinfarction myocardial dysfunction, inhibited AdipoR1 mRNA and protein expression levels. Coincubation of the cells with pravastatin reversed the inhibitory effects of TNF-alpha on AdipoR1 expression. In parallel, pravastatin reversed the TNF-alpha-induced decrease in globular adiponectin-induced 2-deoxy-d-[(3)H]glucose uptake in insulin-treated cultured cells. Moreover, this effect of pravastatin was inhibited by the suppression of AdipoR1 expression by small-interfering RNA specific for AdipoR1. Incubation with H(2)O(2) reduced AdipoR1 expression in cultured cardiomyocytes that were attenuated by N-acetyl-l-cysteine or pravastatin. Pravastatin suppressed TNF-alpha-induced intracellular oxidants in cultured cardiomyocytes. In conclusion, pravastatin reversed the reduction of AdipoR1 expression in postinfarction mouse myocardium and in TNF-alpha-treated cardiomyocytes partly through an antioxidative mechanism in association with improved glucose uptake.     

Different processing of chromogranin B into GAWK-immunoreactive fragments in the bovine adrenal medulla and pituitary gland

Chromogranin B 420-493 (GAWK)-like immunoreactivity (chromogranin B (420-493)-LI) was determined by radioimmunoassay using two different rabbit antisera, one raised against chromogranin B (420-436) (GAWK 1-17) (Ab420-436) and the other against chromogranin B 439-457 (GAWK 20-38) (Ab439-457), in bovine and human tissues. Chromogranin B (420-493)-LI was present in the bovine adrenal medulla chromaffin granules as well as in the anterior pituitary gland and was released from the cultured bovine chromaffin cells by stimulation with high K+ or nicotine. Chromogranin B (420-493)-LI present in the bovine tissues was detected using Ab420-436 but was not detected using Ab439-457. In the human tissues, chromogranin B (420-493)-LI was detected using Ab420-436 as well as Ab439-457. This suggests that the amino acid sequence of this region (chromogranin B 439-457) is different between human and bovine. On the gel permeation chromatography, chromogranin B (420-493)-LI was eluted at the void volume in the bovine adrenal medulla and at an apparent molecular weight of 4000 in the anterior pituitary gland. On the reverse-phase high-performance liquid chromatography, multiple peaks of chromogranin B (420-493)-LI was detected in the bovine adrenal medulla while one component of chromogranin B (420-493)-LI was found in the anterior pituitary gland. These results suggest that chromogranin B is processed into small fragments of chromogranin B (420-493)-LIs and that this processing is tissue-specific.     

Chemical stimulation of the thalamic reticular nucleus inhibits the neuronal activity of the posterior insular cortex in rats

Extracellular neuronal responses were recorded from the posterior insular cortex following electrical and chemical stimulation of the thalamic reticular nucleus (Rt) regions. In the present study, most neurons (29/32) were first characterized for their responses to electrical stimulation of the superior laryngeal (SL) nerve or glossopharyngeal (IXth) nerve. In the first experiment, 15 neurons in the posterior insular cortex were examined for their responses to electrical stimulation of the Rt regions. It was found that effective stimulation sites to evoke action potentials in the posterior insular cortex were the ventromedial portion of the Rt and its adjacent regions. In the second experiment, 17 neurons in the posterior insular cortex were examined for their responses by pressure injection of glutamate (Glu) into the Rt regions. Of the 17 neurons, 13 were inhibited in the spontaneous discharge rate following injection of Glu into the Rt, and the remaining four were unaffected. Histologically, it was demonstrated that Glu injection sites for the case of inhibition were located near or within the Rt. On the other hand, the injection sites for all four non-responsive neurons were located outside of the Rt. These data suggest that excitation of the Rt (GABAergic neurons) causes depression of the neuronal activity in the thalamic relay nucleus and then this may in turn induce depressed neuronal activity in the posterior insular cortex. The results here indicate that neuronal activity in the posterior insular cortex is controlled by the Rt, which has been reported in other sensory systems.     

High SO2 resistance ofClethra barbinervis established in a smoke-polluted area of Ashio,Tochigi Prefecture,Japan

The effect of SO₂ on the photosynthesis ofClethra barbinervis collected from a smoke-polluted area near the Ashio copper smelter in Tochigi Prefecture was compared withC. barbinervis collected from a nonpolluted district in Tsukuba, Ibaraki Prefecture andQuercus mongolica var.grosseserrata grown in a nonpolluted field in Nagano Prefecture. The plants were exposed to 0.5–1.5 p.p.m. SO₂ for 90 min (short-term) and to 0.3 p.p.m. SO₂ for 31–39 days (long-term). TheClethra plants from both sites had a lower intrinsic stomatal conductance and photosynthetic rate thanQuercus plants. Short-and long-term fumigation caused stomatal closure inQuercus plants, but had little effect on the stomatal conductance ofClethra plants. Under short-term fumigation, nonstomatal photosynthetic inhibition per unit of absorbed SO₂ was smallest inClethra plants from Ashio. Long-term fumigation caused photosynthetic decline and visible foliar injury toQuercus plants, but had no effect onClethra plants from Ashio. Consequently,Clethra plants from Ashio had a higher photosynthetic rate thanQuercus plants after long-term fumigation. These results suggest thatC. barbinervis populations in the smoke-polluted area of Ashio had evolved high SO₂ resistance connected with SO₂ detoxification ability in mesophyll cells.     

Association Study of a Functional Variant on ABCG2 Gene with Sunitinib-Induced Severe Adverse Drug Reaction

Sunitinib is a tyrosine kinase inhibitor and used as the first-line treatment for advanced renal cell carcinoma (RCC). Nevertheless, inter-individual variability of drug’s toxicity was often observed among patients who received sunitinib treatment. This study is to investigate the association of a functional germline variant on ABCG2 that affects the pharmacokinetics of sunitinib with sunitinib-induced toxicity of RCC patients in the Japanese population. A total of 219 RCC patients were recruited to this pharmacogenetic study. ABCG2 421C>A (Q141K) was genotyped by using PCR-Invader assay. The associations of both clinical and genetic variables were evaluated with logistic regression analysis and subsequently receiver operating characteristic (ROC) curve was plotted. About 43% (92/216) of RCC patients that received sunitinib treatment developed severe grade 3 or grade 4 thrombocytopenia according to the National Cancer Institute-Common Terminology Criteria for Adverse Events version 3.0, the most common sunitinib-induced adverse reaction in this study. In the univariate analysis, both age (P = 7.77x10^-3, odds ratio (OR) = 1.04, 95%CI = 1.01–1.07) and ABCG2 421C>A (P = 1.87x10^-2, OR = 1.71, 95%CI = 1.09–2.68) showed association with sunitinib-induced severe thrombocytopenia. Multivariate analysis indicated that the variant ABCG2 421C>A is suggestively associated with severe thrombocytopenia (P = 8.41x10^-3, OR = 1.86, 95% CI = 1.17–2.94) after adjustment of age as a confounding factor. The area under curve (AUC) of the risk prediction model that utilized age and ABCG2 421C>A was 0.648 with sensitivity of 0.859 and specificity of 0.415. Severe thrombocytopenia is the most common adverse reaction of sunitinib treatment in Japanese RCC patients. ABCG2 421C>A could explain part of the inter-individual variability of sunitinib-induced severe thrombocytopenia.     

Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation

Background

The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.

Principal Findings

We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.

Conclusions

These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.     

Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.