Correction to "Interleukin-34 promotes the proliferation and epithelial-mesenchymal transition of gastric cancer cells".

[This corrects the article on p. 1968 in vol. 14, PMID: 36310707.].

Impact of job demands on police stress response-the roles of basic psychological needs and job autonomy.

BACKGROUND: Police officers are a high-stress group with special job characteristics, and the Chinese police management system places particularly high demands on police officers. Whether the influence of job demands on officers' job burnout can be deduced to general stress response needs to be verified. Based on the JD-R model, the study aims to explore the impact of job demands on police stress response, whether job autonomy as a job resource has a moderating effect, and whether basic psychological needs mediate this effect. METHODS: A total of 251 police officers in a district-level public security bureau of China, were surveyed using Chinese-language versions of the Job Demands Scale, the Stress Response Scale, the Job Autonomy Scale, and the Basic Psychological Needs Scale. The mediating effect of basic psychological needs and the moderating effect of job autonomy were tested by regression analysis and bootstrap test. RESULTS: Job demands increase police officers' stress response, and job autonomy does not play a buffer role but enhances this impact, and job demands can partially reduce the police stress response through the satisfaction of basic psychological needs, that is, there is a masking effect of basic psychological needs. CONCLUSIONS: Adjusting and optimizing the ratio of job demands and autonomy in police work to provide high guidance under high demands is of great value to reduce the negative stress responses among police officers.

Interleukin-34 promotes the proliferation and epithelial-mesenchymal transition of gastric cancer cells.

BACKGROUND: Interleukin (IL)-34 is a pro-inflammatory cytokine involved in tumor development. The role of IL-34 in the proliferation and epithelial-mesenchymal transition (EMT) of gastric cancer (GC) remains to be investigated. AIM: To investigate whether and how IL-34 affects the proliferation of GC cells and EMT. METHODS: Using immunohistochemical staining, the expression of IL-34 protein was detected in 60 paired GC and normal paracancerous tissues and the relationship between IL-34 and clinicopathological factors was analyzed. The expression of IL-34 mRNA and protein in normal gastric epithelial cell lines and GC was detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Stable IL-34 knockdown and overexpression in AGS cell lines were established by lentiviral infection and validated by qRT-PCR and western blotting. The cholecystokinin-8 assay, clone formation assay, cell scratch assay, and transwell system were used to detect GC cell proliferation, clone formation, migration, and invasion capacity, respectively. The effects of IL-34 on the growth of GC transplant tumors were assessed using a subcutaneous transplant tumor assay in nude mice. The effects of IL-34 on the expression level of EMT-associated proteins in AGS cells were examined by western blotting. RESULTS: Expression of IL-34 protein and mRNA was higher in GC cell lines than in GES-1 cells. Compared to matched normal paraneoplastic tissues, the expression of IL-34 protein was higher in 60 GC tissues, which was correlated with tumor size, T-stage, N-stage, tumor, node and metastasis stage, and degree of differentiation. Knockdown of IL-34 expression inhibited the proliferation, clone formation, migration, and invasion of AGS cells, while overexpression of IL-34 promoted cell proliferation, clone formation, migration, and invasion. Furthermore, the reduction of IL-34 promoted the expression of E-cadherin in AGS cells but inhibited the expression of vimentin and N-cadherin. Overexpression of IL-34 inhibited E-cadherin expression but promoted expression of vimentin and N-cadherin in AGS cells. Overexpression of IL-34 promoted the growth of subcutaneous transplanted tumors in nude mice. CONCLUSION: IL-34 expression is increased in GC tissues and cell lines compared to normal gastric tissues or cell lines. In GC cells, IL-34 promoted proliferation, clone formation, migration, and invasion by regulating EMT-related protein expression cells. Interference with IL-34 may represent a novel strategy for diagnosis and targeted therapy of GC.

A comparison of the efficacy and safety of combined aclidinium bromide and formoterol fumarate in the treatment of chronic obstructive pulmonary disease: A protocol for systematic review and meta-analysis.

BACKGROUND: To systematically evaluate the efficacy and safety of combined aclidinium bromide and formoterol fumarate for chronic obstructive pulmonary disease (COPD). METHODS: Electronic databases including PubMed, MEDLINE, EMBASE, Cochrane Library Central Register of Controlled Trials, WanFang, and China National Knowledge Infrastructure (CNKI) database were searched for studies on the use of combined aclidinium bromide and formoterol fumarate in the treatment of COPD. Two independent researchers performed literature screening, data extraction, and assessment of quality of studies. The strength of the association of the efficacy and safety of combined aclidinium bromide and formoterol fumarate in the treatment of COPD was evaluated according to the odds ratio (OR), mean differences (MDs), and 95% confidence interval (CI). Statistical analysis was carried out via using RevMan 5.3 software. RESULTS: The results of the present study will be published in a peer-reviewed journal. CONCLUSION: The conclusion of the present study will provide evidence to judge whether combined aclidinium bromide and formoterol fumarate is an effective and safety intervention in the treatment of COPD. SYSTEMATIC REVIEW REGISTRATION NUMBER: INPLASY202070063.

Suppression of gastric cancer by extract from the tuber of amorphophallus konjac via induction of apoptosis and autophagy.

The tuber of amorphophallus konjac (TuAK) is an antitumor herb used in traditional Chinese medicine. The present study investigated the inhibitory effect of TuAK against gastric cancer and the underlying mechanisms associated with two programmed cell death pathways, apoptosis and autophagy. TuAK was extracted by organic solvents including ethanol and ligarine. The extract of TuAK, shortened as TuAKe, significantly inhibited the growth of cultured gastric cancer cell lines SGC-7901 and AGS, with IC50 of 35-45 µg/ml. TuAKe could increase cell apoptosis and induce cell cycle arrest. For the apoptosis-associated proteins, expressions of survivin and Bcl-2 were decreased by treatment of TuAKe, and the expression of Bax and caspase-9 was increased. Furthermore, TuAKe could promote autophagy, and the antitumor efficacy of TuAKe was significantly hampered by targeted suppression of autophagy, suggesting that autophagy contributed to TuAKe-induced cell death. Furthermore, patients with gastric cancer who received TuAK-based medicinal decoction achieved improved scores in assessment of life quality compared with those without TuAK treatment. This study demonstrated the antitumor activity of TuAKe against gastric cancer, and is the first report to show that the underlying mechanism is associated with induction of autophagy. Our data provided support of the clinical use of amorphophallus konjac-based medication in combination with classical chemotherapy to achieve optimized outcome for gastric cancer.

Ultrafast dynamics of ligand and substrate interaction in endothelial nitric oxide synthase under Soret excitation.

Ultrafast transient absorption spectroscopy of endothelial NOS oxygenase domain (eNOS-oxy) was performed to study dynamics of ligand or substrate interaction under Soret band excitation. Photo-excitation dissociates imidazole ligand in <300fs, then followed by vibrational cooling and recombination within 2ps. Such impulsive bond breaking and late rebinding generate proteinquakes, which relaxes in several tens of picoseconds. The photo excited dynamics of eNOS-oxy with L-arginine substrate mainly occurs at the local site of heme, including ultrafast internal conversion within 400fs, vibrational cooling, charge transfer, and complete ground-state recovery within 1.4ps. The eNOS-oxy without additive is partially bound with water molecule, thus its photoexcited dynamics also shows ligand dissociation in <800fs. Then it followed by vibrational cooling coupled with charge transfer in 4.8ps, and recombination of ligand to distal side of heme in 12ps.

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177.

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594-604, Δ605-612 and Δ626-634) and the C-terminal tail (Δ14; deletion of 1164-1177). The expression of Δ594-604 and Δ605-612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626-634 and Δ1164-1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594-604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594-604) regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.

[Effects of qingre xiaoji recipe on the migration, chemotaxis, and tube formation capability of human lung adenocarcinoma cell induced human umbilical vein endothelial cells].

OBJECTIVE: To study the effects of Qingre Xiaoji Recipe (QXR) on the migration, chemotaxis, and tube formation capability of human umbilical vein endothelial cells (HUVECs) induced by human lung adenocarcinoma cells (SPC-A-1). METHODS: The QXR groups at different final concentrations (2.5, 5.0, 10.0, and 20.0 mg/mL) and a control group were set up. Then the chemotaxis and tube formation capabilities were observed using Erasion trace test, Transwell chamber assay, and Matrigel matrix tube formation assay. RESULTS: QXR at three concentrations (2.5, 5.0, and 10.0 mg/mL) had inhibition on the migration of HUVECs cultured by SPC-A-1 cell supernatant (P<0.05). Besides, the migration rate decreased along with increased concentrations. The number of chemotactic HUVECs was significantly less than that of the positive control group (P <0.01), and the inhibition rate of chemokine increased along with increased concentrations. Furthermore, the number of chemotactic HUVECs was negatively correlated to the drug concentration (r = -0.830, P <0.01). The area of HUVEC cell tubule formation was significantly lower than that of the positive control group (P <0.01). The inhibition rate of HUVEC cell tubule formation increased along with increased drug concentration. Besides, the area of HUVEC cell tubular formation was negatively correlated with the drug concentration (r = -0.937, P <0.01). CONCLUSION: The mechanism of QXR for fighting against tumor angiogenesis might be correlated with its inhibition on the migration, chemotaxis, and tube formation of tumor vascular endothelial cells.

[Cohort study of 684 pairs of mother-and-child allergic diseases].

OBJECTIVE: To understand allergic diseases related factors in Changzheng Town, Putuo District infants and young children. METHOD: Pregnant women registered in Putuo District, Changzheng Town Community Health Service Center Child Health Clinic within the period from January to December, 2008 were enrolled into this survey, a questionnaire survey. The infants were followed up from birth to 2 years of age. The mother and child survey was conducted for 746 pairs, and 684 pairs had complete data. SAS V9.1 statistical software was used for data processing and statistical analysis. RESULT: The survey showed that prevalence of eczema, allergic rash, and wheezing was 27.9%, 18.9%, and 3.9%, respectively. Multivariate logistic regression analysis showed that allergy in either parent and addition of foods other than milk in infants before 4 months of age were risk factors for eczema; allergy in either parent was also risk factors for allergic rash. Exclusive breastfeeding from birth to 6 months of age was a protective factor for wheezing in infants. Other factors such as parental history of asthma, vitamin supplements to the mothers during pregnancy, mothers' special diet habits, calcium level of infants, etc. had no significant correlation with allergic disorders in infants. CONCLUSION: The risk factors for allergic disorders in infants included allergy in either parent and dietary factors of the infants themselves (prematurely adding other foods). Breastfeeding (for 0 - 6 months of age) was a protective factor for infants' wheezing.

Environment acidity triggers release of recombinant adeno-associated virus serotype 2 from a tunable matrix.

Successful design of a pH responsive polyelectrolyte-based virus delivery matrix with extracellular release triggered by tumor acidosis has been achieved. Recombinant adeno-associated virus serotype 2 (AAV2) is loaded in the polyelectrolyte-based matrix (AAV2-matrix), which is formed by a biodegradable copolymer of poly(polyethylene glycol-1-(3-aminopropyl)imidazole-dl-aspartic acid) with tuned pH response based on inclusion of polyethyleneimine (PEI(800)). Physico-chemical properties of AAV2-matrix are optimized to minimize cellular interactions until a tumor acidosis-like environment protonates the matrix, reverses ζ-potential and causes particles to swell, releasing the AAV2 virus. The pH-dependent release is highly controllable and potentially useful to optimize site specific viral delivery.

Quiescent fibroblasts are more active in mounting robust inflammatory responses than proliferative fibroblasts.

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E(2). As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.

Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding.

Exacerbation of oxidative stress-induced cell death and differentiation in induced pluripotent stem cells lacking heme oxygenase-1.

Embryonic stem cells (ESCs) are promising donor sources in cell therapies for various diseases. Although low levels of reactive oxygen species (ROS) are necessary for the maintenance of stem cells, increased ROS levels initiate differentiation and cell damage. We and others have previously demonstrated that heme oxygenase (HO)-1, a stress response protein with antioxidative and anti-inflammatory properties, plays critical protective functions in cardiovascular and other diseases. However, the functions of HO-1 in ESCs remain to be elucidated. Our goal was to investigate the roles of HO-1 in ESC survival and differentiation. Due to the lack of HO-1-deficient ESCs, we used Oct3/4, Sox2, c-Myc, and Klf4 retroviruses to reprogram mouse embryonic fibroblasts into induced pluripotent stem (iPS) cells of different HO-1 genotypes. These iPS-HO-1 cells exhibited characteristics of mouse ESCs (mESCs) and formed teratomas that were composed of cell types of all 3 germ layers after injected into severe combined immunodeficiency mice. In response to oxidant stress, iPS-HO-1(-/-) cells accumulated higher levels of intracellular ROS compared with D3 mESCs or iPS-HO-1(+/+) cells and were more prone to oxidant-induced cell death. Spontaneous differentiation experiments revealed that Oct4 levels were significantly lower in iPS-HO-1(-/-) cells after leukemia inhibitory factor withdrawal and removal of feeders. Further, during the course of spontaneous differentiation, iPS-HO-1(-/-) cells had enhanced Erk1/2 phosphorylation, which has been linked to ESC differentiation. By the loss-of-function approach using iPS-HO-1(-/-) cells, our results demonstrate that a lack of HO-1 renders iPS cells more prone to oxidative stress-induced cell death and differentiation.

Increased expression of 14-3-3ß promotes tumor progression and predicts extrahepatic metastasis and worse survival in hepatocellular carcinoma.

14-3-3ß is implicated in cell survival, proliferation, migration, and tumor growth; however, its clinical relevance in tumor progression and metastasis have never been elucidated. To evaluate the clinical significance of 14-3-3ß, we analyzed the association of 14-3-3ß expression and clinicopathologic characteristics in primary and subsequent metastatic tumors of hepatocellular carcinoma patients. 14-3-3ß was expressed abundantly in 40 of 55 (70.7%) primary tumors. Increased 14-3-3ß expression in primary tumors predicted a higher 5-year cumulative incidence of subsequent extrahepatic metastasis, and multivariate analysis revealed 14-3-3ß overexpression was an independent risk factor for extrahepatic metastasis. Patients with increased 14-3-3ß expression in primary tumors had worse 5-year overall survival rates, and 14-3-3ß overexpression was an independent prognostic factor on Cox regression analysis. Furthermore, stably overexpressed 14-3-3ß enhanced hepatocellular carcinoma cell migration and proliferation and increased anchorage-independent cell growth. In addition, in vivo study in a nude-mice model showed tumor formation significantly increased with 14-3-3ß overexpression. In conclusion, this is the first report to show that increased 14-3-3ß expression is associated with subsequent extrahepatic metastasis and worse survival rates, as well as cancer progression of hepatocellular carcinoma. Thus, 14-3-3ß may be a novel prognostic biomarker and therapeutic target in hepatocellular carcinoma.

Two synthetic peptides corresponding to the proximal heme-binding domain and CD1 domain of human endothelial nitric-oxide synthase inhibit the oxygenase activity by interacting with CaM.

Human endothelial nitric-oxide synthase (eNOS) is a complex enzyme, requiring binding of calmodulin (CaM) for electron transfer. The prevailing view is that calcium-activated CaM binds eNOS at the canonical binding site located at residues 493-510, which induces a conformational change to facilitate electron transfer. Here we demonstrated that the CaM enhances the rate of electron transfer from NADPH to FAD on a truncated eNOS FAD subdomain (residues 682-1204) purified from baculovirus-infected Sf9 cells, suggesting more complicated regulatory mechanism of CaM on eNOS. Metabolically (35)S-labeled CaM overlay on fusion proteins spanning the entire linear sequence of eNOS revealed three positive (35)S-CaM binding fragments: sequence 66-205, sequence 460-592, and sequence 505-759. Synthetic peptides derived from these fragments are tested for their effects on CaM binding and eNOS catalytic activities. Peptides corresponding to the proximal heme-binding site (E1, residues 174-193) and the CD1 linker connecting FAD/FMN subdomains (E4, residues 729-757) bind CaM at both high Ca(2+) (Ca(2+)CaM) and low Ca(2+) (apoCaM) concentrations, whereas peptide of the canonical CaM-binding helix (E2, residues 493-510) binds only Ca(2+)CaM. All three peptides E1, E2 and E4 significantly inhibit oxygenase activity in a concentration-dependent manner, but only E2 effectively inhibits reductase activity. Concurrent experiments with human iNOS showed major differences in the CaM binding properties between eNOS and iNOS. The results suggest that multiple regions of eNOS might interact with CaM with differential Ca(2+) sensitivity in vivo. A possible mechanism in regulating eNOS activation and deactivation is proposed.

[Impact of serum epidermal growth factor on progressive psoriasis vulgaris and regulation of Chinese herbal medicine for cleaving heat, cooling blood, and detoxicating on it].

OBJECTIVE: To investigate the impact of serum epidermal growth factor (EGF) on progressive psoriasis vulgaris and the regulatory effect of Chinese herbal medicine on it. METHODS: Serum level of EGF was measured in 31 patients with progressive psoriasis vulgaris (the tested group) and 17 healthy subjects (the control group) by ELISA. The changes in EGF level and condition of illness were observed before and after patients orally administered Chinese composite recipe for clearing heat, cooling blood and removing toxic substances for 10 weeks, and the severity of illness was assessed by psoriasis area and severity index (PASI) as well. RESULTS: Serum level of EGF in patients was 175.35 +/- 179.86 ng/L before treatment, markedly higher than that in healthy subjects (72.05 +/- 63.01 ng/L), showing significant difference between them (t = - 2.888, P = 0.006); it reduced to 121.67 +/- 94.74 ng/L after treatment, significantly different to that before treatment (t = 2.155, P = 0.04), but still higher than that in the control (t = - 2.146, P = 0.037). Average PASI was 9.65 +/- 5.82 before treatment and 5.74 +/- 4.69 after treatment, displaying a significant reduction (t = 7.740, P < 0.01). Linear regression analysis showed no apparent correlation between serum EGF level and PASI (r = 0.030, P = 0.872; r = 0.050, P = 0.793). CONCLUSION: Serum level of EGF might be an important factor related to the progress of psoriasis. Chinese herbal medicine for clearing heat, cooling blood and removing toxic substances can lessen the severity of psoriasis, its action in reducing serum EGF is possibly one of the mechanisms for these kinds of herbs in treating psoriasis.

[Effects of ethanol extracts of Panax notoginseng on liver metastasis of B16 melanoma grafted in mice].

OBJECTIVE: To observe the effects of ethanol extracts of Panax notoginseng on the tumor and the liver metastasis in experimental mice grafted with B16 melanoma. METHODS: B16 melanoma was transplanted in the spleen of C57BL/6 mice. The effects of different doses of ethanol extracts of Panax notoginseng on the inhibition rate of spleen tumors and the liver metastasis were observed respectively. RESULTS: The high-, medium-, and low-doses of the extracts and the interferon-alpha (IFN-alpha) can improve the quality of life of the experimental mice. The weights of spleen tumor were lower in the low- and medium-dose extracts-treated groups and the IFN-alpha-treated group than that in the normal saline (NS)-treated group (P<0.05 or P<0.01). The liver metastasis was less in the low- and medium-dose extracts-treated groups and the IFN-alpha-treated group than that in the NS-treated group (P<0.01). CONCLUSION: The ethanol extracts of Panax notoginseng can improve the quality of life of the experimental mice and inhibit the growth of tumor and the liver metastasis.

Mediation of beta-endorphin by ginsenoside Rh2 to lower plasma glucose in streptozotocin-induced diabetic rats.

We investigated the plasma glucose-lowering mechanism(s) of Rh2, a ginsenoside derived from Panax ginseng, in rats with streptozotocin-induced diabetes (STZ-diabetic rats). After intravenous injection over 120 min into fasting STZ-diabetic rats, Rh2 decreased plasma glucose in a dose-dependent manner. In parallel to the lowering of plasma glucose, an increase of plasma beta-endorphin-like immunoreactivity was observed. In addition, naloxone and naloxonazine at doses sufficient to block opioid mu-receptors inhibited the plasma glucose-lowering action of Rh2 in genetically wild-type, diabetic mice. In contrast, Rh2 failed to lower plasma glucose in opioid mu-receptor knockout diabetic mice. An increase in gene expression at both the mRNA and protein levels of glucose transporter subtype 4 (GLUT 4) was observed in soleus muscle obtained from STZ-diabetic rats treated with Rh2 three times daily for one day; this increase in expression was absent when opioid mu-receptors were blocked. In conclusion, our results suggest that ginsenoside Rh2 may lower plasma glucose in STZ-diabetic rats based on an increase in beta-endorphin secretion that activates opioid mu-receptors thereby resulting in an increased expression of GLUT 4.

Structural elements contribute to the calcium/calmodulin dependence on enzyme activation in human endothelial nitric-oxide synthase.

Two regions, located at residues 594-606/614-645 and residues 1165-1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594-606/614-645 resulted in an enzyme (Delta45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155-13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (Delta14) or combined deletions of residues 594-606/614-645 and 1165-1178 (Delta45/ Delta14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Delta45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Delta45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Delta14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2-4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Delta45/Delta14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Delta45/ Delta14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments.

Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on L-arginine hydroxylation and GN-hydroxy-L-arginine oxidation.

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of L-arginine (Arg) with formation of an intermediate, GN-hydroxy-L-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H(4)B and instability of the heme-CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H(4)B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 microM NHA and 0.39 microM H(4)B to reach the half-maximal NHA-driven NO formation rate (EC(50)), while R365L needed 59 microM NHA and 0.73 microM H(4)B to achieve EC(50). The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.