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Fucoidan from Laminaria japonicais an important natural bioactive substance, and low molecular weight fucoidan (L-F) obtained by hydrothermal degradation exhibits better bioavailability. However, the anti-obesity effect of Laminaria japonicafucoidan (FUC) and whether hydrothermal degradation could enhance its activity remained uncertain. The related indicators were therefore determined and compared. According to the results, the molecular weight and glucuronic acid content of FUC were significantly reduced after degradation. Animal experiment results showed that oral administration of 300 mg/kg FUC effectively reduced body weights and serum lipid levels, while these effects of L-F were diminished. Changes in the expression levels of hepatic gene expression showed that FUC inhibited fatty acid synthesis via down-regulation expression of SREBP-1c and FAS, and promoted cholesterol consumption via up-regulation of CYP7A1 and CYP27A1. However, L-F had no significant effect on SREBP-1c and FAS. FUC and L-F also had regulatory effects on gut microbiota, but there were some differences in their specific effects. FUC improved lipid metabolism by regulating gut microbiota such as Colidextribacter, Desulfovibrioand Colidextribacter, so as to play a role in reducing body weight. L-F had weaker regulatory effect on some fat formation related microbes, which resulted in its reduced anti-obesity effect. Summarily, FUC could ameliorate obesity and improve lipid metabolism disorders, but these activities were diminished after hydrothermal treatment, which may connect with the decarboxylation of glucuronic acid and the change in regulatory activity of gut microbiota.

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6AICL3_KGKY) that replaces the ancestral KGRKLP sequence (GPRC6AICL3_RKLP) present in all other species. In vitro assays purport that human GPRC6AICL3_KGKYis retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6AICL3_KGKYin PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY), and humanized mouse (mGPRC6AICL3_KGKY) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6AICL3_KGRKLPacts as a classic G protein–coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6AICL3_KGKYand humanized mouse mGPRC6AICL3_KGKYare retained intracellularly in ligand naive cells, yet exhibit β-arrestin–dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal–regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6AICL3_KGKYis functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6AICL3_KGKY. In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A-deficient cells, the effect of testosterone to activate downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.

To explore the effect of electroacupuncture at conception vessel and governor vessel on correlative signals of inflammatory response stress-injury-repair in cerebral ischemia reperfusion rats.

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic β-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletion of Gprc6ain β-cells (Gprc6aβ-cell-cko) by crossing Gprc6aflox/floxmice with Ins2-Cremice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and β-cell proliferation were reduced in Gprc6aβ-cell-ckocompared with control mice. Gprc6aβ-cell-ckoexhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6aβ-cell-ckomice showed reduced insulin simulation index in response to Ocn. These data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating β-cell proliferation and insulin secretion.

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses of T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca−/−mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.

To investigate the combinatorial effects of conception and governor vessel electroacupuncture (EA) and human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) on pathomorphologic lesion and cellular apoptosis in rats with cerebral ischemia/reperfusion.

BackgroundLong-term anti-depression treatment can promote the regeneration of hippocampal regeneration. Up-regulation of hippocampal regeneration can reverse or prevent against the injury of stress to cerebrum, especially to hippocampal structure and function. Therefore, promoting hippocampal neuronal regeneration may be a new strategy for treating depression and anxiety.ObjectiveTo observe the effect of electro-acupuncture at Du channel and meridian of foot-Taiyang on hippocampal neurons from model rats of depression.DesignA randomized controlled animal experiment.SettingDepartment of Acupuncture and Moxibustion, Shenzhen Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine.MaterialsTwenty-four Wistar rats, of either gender, aged 2 months old, weighing 200–220 g, were provided by the Animal Experimental Center, Guangzhou University of Traditional Chinese Medicine.MethodsThis experiment was carried out in the Clinical Molecular Biochemical Laboratory, Shenzhen Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine between October 2006 and April 2007. ▪The involved rats were randomized into 4 groups according to body mass blank control group, model group, electroacupuncture A group and electroacupuncture B group, with 6 in each. Rats in the blank control group were free to access to water, and were not given any intervention. Rats in the latter 3 groups were developed into rat depression models by chronic stress combined with feeding alone, and received 21-day unpredictable various stresses. Rats in the model group were euthanized at 14 days after modeling, and their brain tissues were harvested. Rats in the electroacupuncture A group were modeled, then points “Baihui” and “Shenting” were chosen, and given electroacupuncture, once a day, 20 minutes once. Rats in the electroacupuncture B group were modeled, then points “Baihui”, “Shenting”, “Xinshu” and “Ganshu” were chosen, and frequency and therapeutic time were the same as those of electroacupuncture A group. Rats in the latter 2 groups were also given electroacupuncture, and their brain tissues were harvested 14 days later. ▪ Index evaluation: Neurons in hippocampal CA1 and CA3 regions as well as dentate gyrus [neuron specific enolase (NSE)-positive cells] were counted by immunofluorescent technique. Each rat was weighted on the day of grouping and before execution at 35 days.Main Outcome MeasuresNeuron amount in each hippocampal region and body mass of rats in each group.ResultsTwenty-four rats were involved in the final analysis. ▪Neuronal amount in each region of hippocampus: Neurons in the hippocampal CA1 and CA3 regions as well as dentate gyrus of rats in the model group were significantly less than those in the blank control group (P < 0.01). Neurons in hippocampal CA1and CA3 regions as well as dentate gyrus of rats in the electroacupuncture A and B groups were more than those in the model group (P < 0.05). ▪ Change in body mass of rats: Body mass increase of rats in the model group was less than that in the blank control group (P < 0.05), and body mass increase of electroacupuncture A and B groups was significantly more than that in the model group, respectively (P < 0.01).ConclusionElectroacupuncture at Du channel and meridian of foot-Taiyang alleviates hippocampal neuronal injury of rats, and thereby, improves depression and promotes the increase of body mass of rats.

Defining the molecular target for strontium in osteoblasts is important for understanding the anabolic effects of this cation on bone. The current studies demonstrate that a G‐protein‐mediated response to strontium persists in osteoblasts that lack CASR, suggesting a predominant role for a novel cation‐sensing receptor in mediating the osseous response to strontium.

Defining the molecular target for strontium in osteoblasts is important for understanding the anabolic effects of this cation on bone. The current studies demonstrate that a G‐protein‐mediated response to strontium persists in osteoblasts that lack CASR, suggesting a predominant role for a novel cation‐sensing receptor in mediating the osseous response to strontium.Introduction:Strontium has anabolic effects on bone and is currently being developed for the treatment of osteoporosis. The molecular target for strontium in osteoblasts has not been determined, but the existence of CASR, a G‐protein‐coupled receptor calcium‐sensing receptor, raises the possibility that strontium actions on bone are mediated through this or a related receptor.Materials and Methods:We used activation of a transfected serum response element (SRE)‐luciferase reporter in HEK‐293 cells to determine if CASR is activated by strontium. In addition, we examined strontium‐mediated responses in MC3T3‐E1 osteoblasts and osteoblasts derived from wild‐type and CASR null mice to determine if other cation‐sensing mechanisms are present in osteoblasts.Results and Conclusions:We found that strontium stimulated SRE‐luc activity in HEK‐293 cells transfected with full‐length CASR but not in cells expressing the alternatively spliced CASR construct lacking exon 5. In contrast, we found that MC3T3‐E1 osteoblasts that lack CASR as well as osteoblasts derived from CASR null mice respond to millimolar concentrations of strontium. The response to strontium in osteoblasts was nonadditive to a panel of extracellular cations, including aluminum, gadolinium, and calcium, suggesting a common mechanism of action. In contrast, neither the CASR agonist magnesium nor the calcimimetic NPS‐R568 activated SRE activity in osteoblasts, but the response to these agonists was imparted by transfection of CASR into these osteoblasts, consistent with the presence of distinct cation‐sensing mechanisms. Co‐expression of the dominant negative Gαq(305–359) minigene also inhibited cation‐stimulated SRE activity in osteoblasts lacking known CASR. These findings are consistent with strontium activation of a novel Gαq‐coupled extracellular cation‐sensing receptor in osteoblasts with distinct cation specificity.

The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression‐cloning strategy, the calcium‐binding protein calcyclin was found to mediate the response of MC3T3‐E1 osteoblasts to extracellular cations, but not the calcimimetic NPS‐568, indicating the presence of another cation‐sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation.

The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression‐cloning strategy, the calcium‐binding protein calcyclin was found to mediate the response of MC3T3‐E1 osteoblasts to extracellular cations, but not the calcimimetic NPS‐568, indicating the presence of another cation‐sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation.Introduction:Extracellular calcium and other cations seem to regulate the function of osteoblasts through a distinct calcium‐sensing mechanism that is coupled to activation of c‐fosgene transcription. The identity of this calcium‐sensing mechanism is unknown.Methods:To identify molecules that participate in this extracellular cation‐sensing pathway, we developed an expression cloning strategy in COS‐7 cells using cation stimulation of a serum response element (SRE) luciferase reporter derived from the c‐fospromoter to screen a mouse MC3T3‐E1 osteoblast cDNA library.Results and Conclusions:We identified calcyclin (S100A6), a calcium‐binding protein of the EF‐hand type belonging to the S100 family, as being responsible for transferring a cation‐sensing response from osteoblasts to COS‐7 cells. Transfection of the calcyclin cDNA into COS‐7 and HEK‐293 cells confirmed that the overexpression of calcylin caused these cells to gain the ability to sense extracellular cations, including aluminum, gadolinium, calcium, and magnesium. Conversely, we found that an antisense calcyclin construct reduced calcyclin expression and partially inhibited the cation‐sensing response in MC3T3‐E1 osteoblasts. These results implicate calcyclin in the activation of SRE and establish a role for calcyclin as an accessory protein involved in the cation‐sensing pathway in osteoblasts.

We isolated osteoblastic cell lines from wild-type (CasR+/+) and receptor null (CasR−/−) mice to investigate whetherCasRis present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from bothCasR+/+and CasR−/−mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasRagonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR−/−osteoblasts. The functional responses to these cations were identical inCasR+/+and CasR−/−osteoblasts. Thus, the absence of CasRalters neither the maturational profile of isolated osteoblast cultures nor their in vitroresponses to extracellular cations. In addition,CasRtranscripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR+/+orCasR−/−osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative forCasRprotein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts fromCasR−/−mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

A new schema proposes that the bone-derived osteocalcin (Ocn) peptide hormone activates the G-protein–coupled receptor GPRC6A to directly regulate glucose and fat metabolism in liver, muscle, and fat, and to stimulate the release of metabolism-regulating hormones, including insulin, fibroblast growth factor 21, glucagon-like peptide 1, testosterone, and interleukin 6. Ocn/GPRC6A activation has also been implicated in cancer progression. GPRC6A is activated by cations, amino acids, and testosterone. The multiligand specificity, the regulation of energy metabolism in diverse tissues, and the coordinated release of metabolically active hormones make the GPRC6A endocrine networks unique. Recently, the significance of Ocn/GPRCA has been questioned. There is a lack of metabolic abnormalities in newly created genetically engineered Ocn- and Gprc6a-deficient mouse models. There are also paradoxical observations that GPRC6A may function as a tumor suppressor. In addition, discordant published studies have cast doubt on the function of the most prevalent uniquely human GPRC6A-KGKY polymorphism. Explanations for these divergent findings are elusive. We provide evidence that the metabolic susceptibility of genetically engineered Ocn- and Gprc6a-deficient mice is influenced by environmental challenges and genetic differences in mouse strains. In addition, the GPRC6A-KGKY polymorphism appears to be a gain-of-function variant. Finally, alternatively spliced isoforms of GPRC6A may alter ligand specificity and signaling that modulate oncogenic effects. Thus, genetic, post-translational and environmental factors likely account for the variable results regarding the functions of GPRC6A in animal models. Pending additional information, GPRC6A should remain a potential therapeutic target for regulating energy and fat metabolism, hormone production, and cancer progression.

Whether the known calcium‐sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti‐CasR antisera (4637) and low stringency reverse transcriptase‐polymerase chain reaction (RT‐PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti‐CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT‐PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a ∼165 kDa and ∼140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild‐type HEK 293 cells or Saos2, MG‐63, or U‐2 OS osteoblast‐like cell lines. Standard two‐step RT‐PCR using CasR‐specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT‐PCR procedure amplified nonspecific products in wild‐type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT‐PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.

Whether the known calcium‐sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti‐CasR antisera (4637) and low stringency reverse transcriptase‐polymerase chain reaction (RT‐PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti‐CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT‐PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a ∼165 kDa and ∼140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild‐type HEK 293 cells or Saos2, MG‐63, or U‐2 OS osteoblast‐like cell lines. Standard two‐step RT‐PCR using CasR‐specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT‐PCR procedure amplified nonspecific products in wild‐type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT‐PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.

Abstract: During the course of mitochondrial DNA sequencing ofDictyostelium discoideum, a sequence with a tRNA-like structure and two genes for tRNAGin(UUG) and tRNATrp(CCA) were found downstream of the gene for large subunit rRNA. The existence of tRNATrp with CCA anticodon supports the finding that UGA codon is not a tryptophan codon inD. discoideum mitochondria. Interestingly, the tRNA gene-like sequence (t-element) has TTA at the anticodon position. Northern blot analysis showed that, in low molecular mass mitochondrial RNA fraction of growth-phase cells and developmental stage cells, a mature transcript of the ele ment could not be detected in the tRNA region on an ureadenatured 15% polyacrylamide gel, although there were several bands in the higher molecular mass region, indicating the actual transcription of the t-element. Southern blot analysis for total and mitochondrial DNA showed that the element exists as a single copy, only in the mitochondrial DNA but not in the nuclear DNA.

IntroductionDuring the process of caring for patients, nursing attendants frequently require to help patients with lifting/transferring, patting/turning and rehabilitation. Nursing attendants are required to exert forceful and awkward postures for extended periods of time that caused musculoskeletal disorders. This study is to survey the work situation, physiological workloads, and musculoskeletal disorders associated with prolonged nursing attendance tasks in nursing attendants.MethodThe self-administered questionnaires are assessed via a cross-sectional study of 190 female workers in Taiwan. Information is obtained on demographics, job characteristics, health status, and physiological workload.Results and DiscussionThe observational result shows that the most common prevalence of physical discomfort was lower back (69.5%), followed by right shoulder (47.9%), left shoulder (44.2%), and neck (37.9%). Meanwhile, the most pronounced tired is to help patients with lifting/transferring (79.2%), followed by patting/turning (55.1%). The anticipated results of this study could be a workplace task design reference for improvement of musculoskeletal fatigue and disorders among nursing attendants.