Glucocorticoids cause secondary osteoporosis and myopathy. The efficacy of vitamin D on osteoporotic fractures is thought to be through direct effects on bone and indirect effects on muscles that help to prevent falls. However, effects of vitamin D on muscles under glucocorticoid treatment remain unclear. Six-month-old female Wistar rats were randomized to four groups: vehicletreated controls; a prednisolone (PSL)-administered group (PSL group); an alfacalcidol-administered group (D group); and a group administered both PSL and alfacalcidol (PSL+D group). After a 4-week treatment period, maximum contractile strength and strength decrement index (SDI), an indicator of muscle fatigue, were measured in the calf muscle by electrical stimulation of the sciatic nerve. Cross-sectional area (CSA) of muscle fibers in the tibialis anterior muscle and bone mineral density (BMD) of the femur were evaluated. The PSL group showed significantly lower muscle strength, BMD and CSA of muscle fibers, and significantly higher SDI compared to the other three groups (P < 0.05). No significant differences were observed in any of these parameters among control, D, and PSL+D groups. These results suggest that in glucocorticoid-treated rats, alfacalcidol preserved not only BMD, but also muscle strength and muscle volume, and prevented muscle fatigue.
We have previously demonstrated that vascular endothelial growth factor (VEGF) is critical for cerebral angiogenesis in middle-aged female rats and may play a role in the flow-preserving neuroprotective actions of estrogen through its angiogenic and antiapoptotic properties. Here, we attempt to elucidate the effects of estrogen and the specific estrogen receptor (ER) subtype in cerebral VEGF/Akt/NO pathways and cerebral angiogenesis using 15-week old female mice that are either wild-type (WT), lack estrogen receptor α (ERαKO) or β (ERβKO). Protein levels of VEGF and basic signaling molecules of VEGF angiogenic pathway in the frontal cortex were expressed as follows, as revealed by ELISA and immunoblotting : a) VEGF; WT: ERαKO: ERβKO, 47 ± 15: 27 ± 5: 28 ± 5 pg/mg, respectively (P < 0.01); b) KDR decreased about 40% in both ERαKO and ERβKO compared to WT; c) Akt was significantly down regulated in both ERαKO and ERβKO comp red to WT; d) phosphorylated Akt (pAkt); WT: ERαKO: ERβKO, 0.6 ± 0.2: 0.3 ± 0.01: 0.3 ± 0.1 units/mg, respectively; e) phosphorylated eNOS significantly decreased about 45% in both ERαKO and ERβKO compared to WT. Cerebral capillary density decreased in both ERαKO and ERβKO compared to WT. Thus, it can be concluded that in female mice VEGF/Akt/eNOS pathway plays an important role in cerebral angiogenesis and that both ER subtypes are involved in the regulation of VEGF and its signaling molecule expression in the frontal cortex.
Fish protein is a source of animal protein that is consumed worldwide. Although it has been reported that the intake of Alaska pollack protein (APP) reduces serum triglyceride and body fat accumulation in rats, the mechanisms underlying these effects are poorly understood. In the present study, we fed 5-week-old male Sprague-Dawley rats a high-fat diet with APP or casein for 4 weeks. We reconfirmed that the intake of APP decreases serum triglycerides and inhibits visceral body fat accumulation in rats. We found that APP had a higher non-digestive protein content than casein, and the amount of protein in feces was higher in the APP group than in the casein group. However, the amount of total lipids in feces did not differ significantly between the groups. We also found that the gastrocnemius muscle, a fast-twitch muscle, tended to increase in weight, and that the epididymal fat weight correlated negatively with gastrocnemius muscle weight in the APP group. These results imply that the enhancement of basal energy expenditure by fast-twitch muscle hypertrophy, rather than the enhancement of lipid excretion via feces, partly causes APP-induced inhibition of lipid accumulation in rats.
Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.
Sepsis is induced by infectious challenges, and septic organ failure often occurs under local and systemic inflammation. Albuminuria is also evident during sepsis, but little is known about the molecular basis of septic albuminuria. Using lipopolysaccharide (LPS)-treated mice as a sepsis model, we found that the loss of nephrin, a key component for maintaining podocyte slit diaphragm, became evident in accordance with the onset of albuminuria, especially 36 h post-LPS challenge (i.e., albumiuric stage). Likewise, nephrin mRNA levels were decreased to 13% of saline-treated mice. Such a transcriptional suppression of nephrin was associated with the loss of nucleus-localized Wilms tumor-1 (WT1), a transcriptional factor for up-regulating nephrin gene. Thereafter, urinary albumin levels were decreased in mice between 72 and 96 h post-LPS challenge (i.e., recovery-stage). Notably, nuclear localization of WT1 seemed to be normalized, and nephrin mRNA and protein levels returned near the basal level 72 h post-LPS challenge. During LPS-mediated sepsis, there was a transient increase in blood interleukin-1β, a suppressor of nephrin production in podocytes. Therefore, down-regulation of nephrin by the loss in nuclear WT1, along with hyper-cytokinemia, may underlie the mechanisms by which albuminuria is induced by infectious stresses.
We previously established stable transformants of the human embryonic kidney 293 (HEK293) cell line that express type IV collagen α3, α4 and wild-type or mutant-type α5 chains. Using these cell lines, we confirmed that these three chains form a heterotrimer and that α5 chains containing mutations seen in Alport syndrome are defective in heterotrimerization. In these studies, the amount of heterotrimer that formed was much less than expected relative to the amount of α(IV) chains expressed. The aim of the present study was to determine the effect of the collagen-specific molecular chaperone heat shock protein 47 (HSP47), whose expression is low in HEK293 cells, on the heterotrimerization of α3(IV), α4(IV) and α5(IV) chains. Reduction of HSP47 levels by siRNA resulted in defects of heterotrimerization among the three chains, indicating that HSP47 plays a critical role in the heterotrimerization. On the other hand, overexpression of HSP47 did not influence heterotrimerization. Since many enzymes and molecular chaperons assist correct folding and trimerization of collagens, one or more enzymes and/or molecular chaperones, other than HSP47, might be deficient in HEK293 cells. Overexpression of HSP47 decreased the secretion of heterotrimers containing the mutant α5(IV) chain, suggesting that HSP47 overexpression might enhance the quality control mechanisms of collagen synthesis by inhibiting the secretion of incorrectly structured heterotrimers.
We presently found that medium-chain fatty acids (MCFAs) with 8-12 carbons and their esters facilitated activation (phosphorylation) of mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) 1/2 of cultured embryonic cortical/hippocampal neurons. In particular, trans-2-decenoic acid ethyl ester (DAEE) had the most potent activity. Additionally, DAEE activated phosphatidylinositol 3-kinase and cAMP-response element binding protein (CREB), suggesting that DAEE generates similar intracellular signal as neurotrophins. Therefore, details of the signal elicited by DAEE were examined in comparison with those of a neurotrophin, brain-derived neurotrophic factor (BDNF). We found that 1) DAEE phosphorylated MAPK/ERK1/2 via MEK activation without the involvement of tyrosine kinases of neurotrophin Trk receptors; 2) DAEE activated CREB predominantly through MAPK/ERK1/2 activation, not through other pathways such as cAMP/protein kinase A; and 3) DAEE increased the expression of RNAs of BDNF and neurotrophin-3 and the protein content of synapse-specific proteins such as synaptophysin, synapsin-1, and syntaxin. Based on these observations we propose that DAEE and some other derivatives of MCFAs having neurotrophin-like neurotrophic activities may become therapeutic tools for certain neurological or psychiatric disorders.
The protein components of saliva reflect the condition of the whole body as well as the salivary glands. The aim of this study is to characterize the gene expression profiles in each of the rat major salivary glands-the parotid, submandibular, and sublingual glands. Gene expression was analyzed using DNA microarrays, and observed differences in expression of representative genes were confirmed by quantitative, real-time polymerase chain reaction. Among the glands, the contribution to the high expression of genes encoding various proteins, specifically mucin 10, proline-rich glycoproteins, proline-rich protein 2, proline-rich proteoglycans, cystatin 10, amylase, deoxyribonuclease I, and von Ebner's gland protein, was significantly greater in the parotid gland than the other glands. The submandibular and sublingual glands had similar gene expression profiles that differed from profile of the parotid gland. For example, the genes encoding mucin 19 and ovomacroglobulin were highly expressed only in the submandibular and sublingual glands. In summary, we characterized gene expression in the rat major salivary glands and provided basic information on salivary gland marker proteins.
More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had ‘gain-of-function (GOF)’ activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutantions occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.
OSU03012, a novel celecoxib derivative, has been shown to inhibit proliferation and induce apoptosis in numerous cancer cell lines. However, not much is known about its influence on cell volume regulation and cardiac function in the mammalian heart. We examined the effects of OSU-03012 on cell volume and action potentials in mouse ventricular cells. Video image analysis showed that cell volume increased on application of OSU-03012 in a dose-dependent manner. The action potential duration (APD) at 50% and 90% repolarization (APD50 and APD90 respectively) as well as the resting membrane potential (RMP) were measured in current-clamp experiments. OSU-03012 had little effect on APD50 and RMP but induced approximately 30% shortening of APD90. These results for cell volume and AP are similar to those in cells under ischaemia/hypoxia, and we confirmed that the shortening of APD90 was almost completely recovered by glibenclamide, a potent inhibitor of ATP-sensitive potassium channels.We concluded that OSU-03012 may lead to cell swelling and shortening of AP via reduced ATP production in mouse ventricular cells.