[Effect of naringin on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy].

To explore the protective effect of naringin(Nar) on the injury of myocardium tissues induced by streptozotocin(STZ) in diabetic rats and the relationship with oxidative stress and endoplasmic reticulum stress(ERS)， the male SD rats were intraperitoneally injected with streptozotocin(STZ， 60 mg·kg⁻¹) to establish the diabetic rat model and then randomly divided into the type 1 diabetic rat group(T1DR)， the low-dose Nar group(Nar25)， the middle-dose Nar group(Nar50) and the high-dose Nar group(Nar100). The normal rats were designed as control group(Con). Nar25， Nar50， Nar100 groups were orally administered with Nar at the doses of 25.0， 50.0， 100.0 mg·kg⁻¹ per day， respectively， while the normal group and the T1DR group were orally administered with saline. At the 8th week after treatment， fasting plasma glucose and heart mass index were measured. The pathological changes in myocardial tissues were observed by microscope. The cardiac malondialdehyde(MDA) level and superoxide dismutase(SOD) activities were measured. The gene and protein expressions of glucose-regulated protein 78(GRP78)， C/EBP homologous protein(CHOP)， cysteinyl aspartate-specific proteinase 12(caspase 12) were detected by qRT-PCR and Western blot. According to the results， compared with control group， the myocardial structure was damaged， the content of MDA was increased， while the activities of SOD were decreased(P<0.05) in T1DR group. GRP78， CHOP and caspase 12 mRNA and protein expressions were increased significantly in T1DR group(P<0.05， P<0.01). Compared with T1DR group， myocardial structure damage was alleviated in Nar treatment group. The content of MDA was decreased， while the activities of SOD were increased significantly. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(P<0.05， P<0.01). After treatment with Nar for 8 weeks， myocardial structure damage was obviously alleviated in Nar treatment groups. The content of MDA was decreased， while the activities of SOD were increased significantly in myocardial tissues. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(P<0.05， P<0.01). The findings suggest that Nar may protect myocardium in diabetic rats by reducing mitochondrial oxidative stress injuries and inhibiting the ERS-mediated cell apoptosis pathway.

MicroRNA-23b Targets Ras GTPase-Activating Protein SH3 Domain-Binding Protein 2 to Alleviate Fibrosis and Albuminuria in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a frequent and severe complication of diabetes that is structurally characterized by glomerular basement membrane thickening, extracellular matrix accumulation, and destabilization of podocyte foot processes. MicroRNAs (miRNAs) are dysregulated in DN, but identification of the specific miRs involved remains incomplete. Here, we confirm that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of miR-23b compared with those of their nondiabetic counterparts. Furthermore, exposure to high glucose downregulated miR-23b in cultured kidney cells. In contrast, renal expression of Ras GTPase-activating protein SH3 domain-binding protein 2 (G3BP2), a putative miR-23b target, increased in DN. In vitro, overexpression of miR-23b decreased, and inhibition of miR-23b increased, G3BP2 expression levels. Bioinformatics analysis also revealed p53 binding sites in the miR-23b promoter; in vitro inhibition of p53 or the upstream p38 mitogen-activated protein kinase (p38MAPK) upregulated miR-23b expression in high-glucose conditions. In turn, inhibition of G3BP2 or overexpression of miR-23b downregulated p53 and p38MAPK expression in high-glucose conditions. In vivo, overexpression of miR-23b or inhibition of p53 in db/db mice reversed hyperalbuminuria and kidney fibrosis, whereas miR-23b antagomir treatment promoted renal fibrosis and increased albuminuria in wild-type mice. These data suggest that hyperglycemia regulates pathogenic processes in DN through an miR-23b/G3BP2 feedback circuit involving p38MAPK and p53. In conclusion, these results reveal a role for miR-23b in DN and indicate a novel potential therapeutic target.

Nicotine inhibits microglial proliferation and is neuroprotective in global ischemia rats.

Ischemic injury in rodent models reliably leads to the activation of microglia, which might play a detrimental role in neuronal survival. Our preliminary studies suggest that nicotine plays a potential role in decreasing the numbers of cultured microglia in vitro. In the present study, we found treatment with nicotine 2, 6, and 12 h after ischemia for 7 days significantly increased the survival of CA1 pyramidal neurons in ischemia/reperfusion rats. This effect was accompanied by a significant reduction in the increase of microglia rather than astrocytes, as well as a significant reduction of enhanced expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) in CA1 induced by ischemia/reperfusion. Nicotine inhibits microglial proliferation in primary cultures with and without the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). Pre-treatment with α-bungarotoxin, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) antagonist, could prevent the inhibitory effects of nicotine on cultured microglial proliferation suggesting that nicotine inhibits the microglial proliferation in an α7 nAChR-dependent fashion. Our results suggest that nicotine inhibits the inflammation mediated by microglia via α7 nAChR and is neuroprotective against ischemic stroke, even when administered 12 h after the insult. α7 nAChR agonists may have uses as anti-ischemic compounds in humans.

Amygdaloid corticotropin-releasing factor is involved in the anxiolytic effect of acupuncture during ethanol withdrawal in rats.

In a previous study, acupuncture at acupoint HT7 attenuated ethanol withdrawal-induced anxiety-like behavior in rats by normalizing amygdaloid catecholamines. In the present study, the involvement of amygdaloid corticotropin-releasing factor (CRF) in the anxiolytic effect of acupuncture was investigated during ethanol withdrawal. Rats were intraperitoneally treated with 3 g /kg/day of ethanol for 28 days, and the CRF mRNA levels in the central nucleus of the amygdala (CEA) were measured by using a RT-PCR analysis 72 hours after the last dose of ethanol. During ethanol withdrawal, the rats were bilaterally treated with acupuncture at acupoints HT7, PC6 or at a non-acupoint (Tail) for one min/day for three days. Also, rats were bilaterally injected with CRF into the CEA five minutes after the third acupuncture treatment, after which followed by the elevated-plus maze (EPM) test and the plasma corticosterone radioimmunoassay (RIA) were administered. The RT-PCR analysis showed a significant increase in the amygdaloid CRF mRNA levels in the ethanol-withdrawn rats compared with both the saline-treated rats and the rats treated with acupuncture at HT7, but neither acupuncture at PC6 nor acupuncture at a non-acupoint significantly inhibited the increased mRNA expression. The EPM test and the RIA also showed that the post-acupuncture infusion of CRF greatly reduced the anxiolytic effect of acupuncture at HT7. These results suggest that during ethanol withdrawal, the anxiolytic effect of acupuncture may be mediated through the modulation of amydaloid CRF during ethanol withdrawal.

A dynamic interplay between alternative polyadenylation and microRNA regulation: implications for cancer (Review).

Alternative polyadenylation and microRNA regulation are both mechanisms of post-transcriptional regulation of gene expression. Alternative polyadenylation often results in mRNA isoforms with the same coding sequence but different lengths of 3' UTRs, while microRNAs regulate gene expression by binding to specific mRNA 3' UTRs. In this sense, different isoforms of an mRNA may be differentially regulated by microRNAs, sometimes resulting in cellular proliferation and this mechanism is being speculated on as a potential cause for cancer development.

[Involvement of inhibition of nucleus GAPDH over-expression in erythropoietin's reduction of neuronal apoptosis induced by brain ischemia/reperfusion in rats].

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.

Expression of TRAIL, DR4, and DR5 in bladder cancer: correlation with response to adjuvant therapy and implications of prognosis.

OBJECTIVES: To explore the interrelationship of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors DR4 and DR5 expressions level with patient prognosis and the response to adjuvant therapy in bladder cancer, the synergism function that is between chemotherapy and TRAIL on apoptosis induction in tumor cells. METHODS: The expression of TRAIL, DR4, and DR5 was studied using immunohistochemistry of paraffin-embedded tumor specimens from 229 bladder cancer patients who had undergone transurethral resection. RESULTS: Cytoplasmic TRAIL, DR4, and DR5 expressions were detected in 35%, 75.1%, and 74.2% of bladder cancer patients, respectively. Patients with bladder cancer with either high DR4 or DR5 expression had a significantly longer postoperative recurrence-free rate than those with low expression of both during the 10-year follow-up. Multivariate analysis revealed that the expression of DR4 (P < .001), DR5 (P < .001) and epirubicin therapy (P = .034) were independent prognostic indicators of bladder cancer. Furthermore, epirubicin therapy significantly improved recurrence-free rate for the patients with DR4-high (P = .006) or DR5-high (P = .042) tumor. CONCLUSIONS: The results of the present study have shown for the first time that a combination of DR4 and DR5 expression have significant value in predicting the prognosis of bladder cancer. In addition, patients with high expression of both DR4 and DR5 might benefit from epirubicin therapy.

[Effect of Schwann cells on differentiation of rat bone marrow mesenchymal stem cells at different ages].

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) are multipotent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and oligodendrocytes in rats at different ages in vitro. METHODS: SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myelin basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregulin 1 (NRG1) was detected by ELISA method. RESULTS: SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or oligodendrocytes-like morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% +/- 2.86%, 12.89% +/- 1.78%, and 2.69% +/- 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% +/- 2.39%, 6.33% +/- 1.40%, and 0.92% +/- 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P < 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P < 0.05). CONCLUSION: The microenvironment formed by SCs can promote BMSCs differentiation into neurons and oligodendrocytes, but the differentiation capability of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is likely important factors that induce the differentiation of BMSCs into neurons and oligodendrocytes.

Icariin induces mouse embryonic stem cell differentiation into beating functional cardiomyocytes.

Icariin, the primary active component of Epimedium extracts, has recently been shown to induce cardiomyocyte differentiation of murine embryonic stem (mES) cells in vitro. However, as these cardiomyocytes were not functionally characterized, the potential application of icariin-induced cardiomyocytes in clinical practice remains unclear. Therefore, in this study, we characterized the structure and function of icariin-induced cardiomyocytes to evaluate their potential application in transplantation for cardiac failure treatment. mES cells were cultured as embryoid bodies (EBs) via the direct suspension method in the presence of icariin. The protein expression profiles and ultrastructural characteristics of mES cell-derived cardiomyocytes were then characterized by immunofluorescence and transmission electron microscopy, respectively. In addition, the expression of cardiac-specific and calcium handling genes was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Cardiomyocytes induced by icariin treatment expressed the cardiac-specific proteins myosin light chain-1v (MLC1v), atrial natriuretic polypeptide (ANP), and cardiac troponin I (cTnI). Furthermore, these cells appeared to possess myofibrils organized into mature sarcomeres that had formed A and I bands. In addition, icariin treatment upregulated the mRNA levels of MLC1v, ANP, cTnI, calsequestrin (CSQ), and sodium-calcium exchanger (NCX) in these cells. Icariin induces the differentiation of mES cells into beating cardiomyocytes with normal structure and function. Therefore, these cells may have promising applications in cardiac cell therapy or tissue engineering.

Functional and histological improvement of the injured spinal cord following transplantation of Schwann cells transfected with NRG1 gene.

In this study, we implanted Schwann cells (SCs) transfected with Neuregulin 1 (NRG1) gene into rats with hemisection spinal cord injury, determined its effects on the repair of spinal cord injury and investigated the underlying mechanisms. Primary SCs were cultured, purified, and transfected with NRG1 gene. SCs and SCs transfected with NRG1 gene were implanted, respectively, into rats with hemisection spinal cord injury. Behavior, imaging, electrophysiology, and immuno-histological analyses were performed to evaluate the effect of NRG1 gene-transfected SCs on the repair of spinal cord injury. In vitro studies showed that NRG1 protein was highly expressed in SCs transfected with NRG1 gene. In addition, the receptors for NRG1, ErbB2, and ErbB4, were upregulated in a time-dependent manner. NRG1-transfected SCs secreted large amount of NRG1 proteins in vivo, which efficiently promoted the expression of ErbB2 and ErbB4 in the neurons and neuroglia cells. Moreover, the number of NSE- and GFAP-positive cells was increased. After cell transplantation, many transplanted cells survived and migrated to the areas with spinal cord injuries. The injuries were recovered in all the experimental groups, but the most significant recovery was observed in the group of rats implanted with SCs transfected with NRG1 gene. We conclude that NRG1-transfected SCs can significantly increase the effect on the repair of spinal cord injury. This repair effect is achieved via the upregulation of ErbB receptor in the target cells, increased proliferation of glial cells, and protection of neurons from apoptosis.

Inhibition of human and rat 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances.

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3beta-HSD activity with IC(50) of 1.35 + or -0.05 and 1.77 + or - 0.04 microM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 microM. All chemicals tested weakly inhibited human 3beta-HSD activity with IC(50)s over 250 microM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17beta-HSD3 activity with IC(50)s of 6.02 + or - 1.02, 4.39 + or - 0.46 and 127.60 + or - 28.52 microM, respectively. The potencies for inhibition of 17beta-HSD3 activity were determined to be PFOSK>PFOS>PFOA>PFHxSK=PFBSK for human 17beta-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC(50)s of PFOS(K) for inhibition of rat 17beta-HSD3 activity was greater than 250 microM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3beta-HSD and human 17beta-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.

YeastWeb: a workset-centric web resource for gene family analysis in yeast.

BACKGROUND: Currently, a number of yeast genomes with different physiological features have been sequenced and annotated, which provides invaluable information to investigate yeast genetics, evolutionary mechanism, structure and function of gene families. DESCRIPTION: YeastWeb is a novel database created to provide access to gene families derived from the available yeast genomes by assigning the genes into putative families. It has many useful features that complement existing databases, such as SGD, CYGD and Génolevures: 1) Detailed computational annotation was conducted with each entry with InterProScan, EMBOSS and functional/pathway databases, such as GO, COG and KEGG; 2) A well established user-friendly environment was created to allow users to retrieve the annotated genes and gene families using functional classification browser, keyword search or similarity-based search; 3) Workset offers users many powerful functions to manage the retrieved data efficiently, associate the individual items easily and save the intermediate results conveniently; 4) A series of comparative genomics and molecular evolution analysis tools are neatly implemented to allow users to view multiple sequence alignments and phylogenetic tree of gene families. At present, YeastWeb holds the gene families clustered from various MCL inflation values from a total of 13 available yeast genomes. CONCLUSIONS: Given the great interest in yeast research, YeastWeb has the potential to become a useful resource for the scientific community of yeast biologists and related researchers investigating the evolutionary relationship of yeast gene families. YeastWeb is available at http://centre.bioinformatics.zj.cn/Yeast/.

Coal dust contiguity-induced changes in the concentration of TNF-alpha and NF-kappa B p65 on the ocular surface.

To observe the influence of coal dust on ocular surface of coal miners and rabbits with coal dust contiguity on expression TNF-alpha and NF-kappa Bp65 and dry eye occurrence. Tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Expression TNF-alpha and NF-kappa Bp65 in ocular surface were determined. Results showed tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Coal dust exposure was linked to development of xerophthalmia, and induced a higher expression of NF-kappa B p65 and TNF-alpha perhaps as a mechanism to resist coal dust ocular surface injury.

Thymidylate synthase was associated with patient prognosis and the response to adjuvant therapy in bladder cancer.

OBJECTIVE: To investigate the expression of thymidylate synthase (TS), a key enzyme in DNA synthesis that is over-expressed in several cancer cells, in bladder cancer and its association with patient prognosis and the response to adjuvant therapy. PATIENTS AND METHODS: In all, 67 bladder tissue specimens were obtained from patients who had undergone transurethral resection (TUR). TS expression in bladder cancer and normal bladder tissue was analysed by immunohistochemistry. RESULTS: Of the 67 bladder tissue specimens, 47 (70%) and 10 (15%) had positive expression for TS in cancer and normal tissues, respectively. TS expression was greater in patients with Grade 3 (16/17, 94%) than in Grade 1 and 2 (31/50, 64%; P = 0.002). It was also greater in Stage T1 (14/14) than in Stage Ta (33/53, 62%; P = 0.001). Furthermore, patients with negative TS expression had a longer postoperative recurrence-free survival (RFS) than those with positive expression during the 5 year follow-up (P = 0.028). In the patients with positive TS-expressing tumours, adjuvant therapy significantly improved RFS (P < 0.001). CONCLUSIONS: High TS expression might be a marker of poor prognosis for patients with bladder cancer. In addition, patients with high TS expression might also be benefit from adjuvant therapy.

An insertion of intracisternal A-particle retrotransposon in a novel member of the phosphoglycerate mutase family in the lew allele of mutant mice.

Intracisternal A-particle retrotransposons (IAPs) are known, moveable, retrovirus-like elements and are defective in envelope protein synthesis in the mouse genome. Insertion of IAP elements can either interupt or enhance gene function or expression. Using a mouse model called lethal wasting (lew), we recently identified the insertion of an IAP sequence in a gene, 9630033F20Rik, that contains domains involved in glycolysis. The expression pattern of the 9630033F20Rik gene between various normal and diseased tissues was determined by semi-quantitative RT-PCR. The effect of the insertion mutation in 9630033F20Rik on glycolysis in heart, muscle, and brain tissues was further investigated using oligonuleotide microarray analysis. Results indicated that the expression of 9630033F20Rik is ubiquitous and its signal is relatively higher in heart and brain tissues. The insertion caused the deletion of exon 5 and decreased expression of this gene in all the tissues studied in the lew mice. Changes in the expression levels of glycolytic genes mainly occured in muscle tissue, raising a possibility that 9630033F20Rik may function as one of the transcriptional regulators of glycolytic genes in skeletal muscle. However, considering the fact that a single nucleotide mutation in vesicle-associated membrane protein 1 (VAMP1) has been reported as the causal gene for the lew mouse, how much of an impact the IAP insertion in the lew mouse phenotype has on glycolytic genes compared to the effect from the VAMP1 mutation responsible for the lew mouse phenotype should be further investigated.

[Effect of Rehmannia glutinosa Libosch water extraction on gene expression of proinsulin in type 2 diabetes mellitus rats].

OBJECTIVE: To investigate the mechanisms of treating 2-DM by Rehmannia glutinosa Libosch water extraction (RGLE). METHODS: The mRNA level of proinsulin in rats panreas tissue was examined by semi-quatitativa RT-PCR,and the protein was measured by SDS-PAGE. RESULTS: The mRNA and protein expressions of proinsulin in RGLE group were higher than those of diabetic model group (P<0.01). The levels of FPG decreased. FINS,IS, HbetaCI increased (P<0.01). CONCLUSION: It may be the mechanism how the RGLE to decline high FPG and cure the 2-DM.

A deletion mutation in Slc12a6 is associated with neuromuscular disease in gaxp mice.

Giant axonopathy (gaxp), an autosomal recessive mouse mutation, exhibits ataxia of the hind legs with a slight side-to-side wobble while walking. Within the genomic region of the gaxp locus, a total of 94 transcripts were identified; the annotation of these genes using OMIM and PubMed yielded three potential candidate genes. By cDNA microarray analysis, 54 genes located on or near the gaxp locus were found to exhibit differential expression between gaxp and littermate controls. Based on microarray data and the known function of genes identified, Slc12a6 was selected as the primary candidate gene and analyzed using the Reveal technology of SpectruMedix. A 17-base deletion was detected from within exon 4 of Slc12a6. Reverse transcriptase polymerase chain reaction validated the difference in Slc12a6 expression in different types of mice at the mRNA level, revealing a marked reduction in gaxp mice. Western blot analysis indicated that the protein product of Slc12a6, the K(+)-Cl(-) cotransporter Kcc3, was not detectable in gaxp mice. The causative role of the exon 4 mutation within Slc12a6 in the gaxp phenotype was further confirmed by screening multiple inbred strains and by excluding the mutation of nearby genes within the gaxp locus.

[Phentolamine antagonizes the effects of norepinephrine on the activity of pain-related neurons in the parafascicular nucleus of morphine-dependent rats].

OBJECTIVE: To examine the antagonization of phentolamine against the effects of norepinephrine (NE) on the activity of pain-related neurons in the parafascicular nucleus of morphine-dependent rats. METHODS: Electric impulses were applied as nociceptive stimulus to the right sciatic nerve of morphine-dependent rats, and the discharges of the pain-related neurons in the parafascicular nucleus were recorded by extracellular recording method with glass microelectrodes. RESULTS: Intracerebroventricular injection of norepinephrine resulted in the inhibition of evoked response of the pain-excited neurons as well as the excitation of evoked response of the pain-inhibiting neurons. Both the inhibitory effect on the electric discharges of the pain-excited neurons and the excitatory effect on the pain-inhibiting neurons of norepinephrine were almost completely blocked by intracerebroventricular administration of phentolamine. CONCLUSION: Phentolamine antagonizes the inhibitory effect of norepinephrine on the activity of pain-related neurons in the parafascicular nucleus in morphine-dependent rats, and norepinephrine may play an important role in the integration of the pain signal through the alpha-receptors.

Inhibitory effect of schisandrin B on gastric cancer cells in vitro.

AIM: To investigate the inhibitory effect and possible mechanism of action of schisandrin B in SC-B on gastric cancer cells in vitro. METHODS: SC-B consisted of schisandrin B, aloe-emodin, and Astragalus polysaccharides. Exponentially growing human gastric cancer SGC-7901 cells were divided into six treatment groups: (1) control group (RPMI 1640 medium); (2) negative control group (2% DMSO); (3) positive control group (50 mg/L 5-Fluorouracil, 5-FU); (4) low-dose group (LSC, final concentration of schisandrin B, 25 mg/L); (5) moderate-dose group (MSC, final concentration of schisandrin B, 50 mg/L); (6) high-dose group (HSC, final concentration of schisandrin B, 100 mg/L). Follow-up was done at 12-48 h. An MTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay was used to examine the inhibitory effect of SC-B on gastric cancer cells. The mitosis index was assessed using an inverted microscope. Flow cytometry was used to visualize the cell cycle. An RT-PCR (Reverse transcription-Polymerase chain reaction) -based assay was used to detect mRNA expression for cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The MTT assay showed that the number of living cells in the LSC, MSC and HSC groups was significantly smaller than that in the DMSO-treated group (P < 0.05) at 12-48 h. The inhibitory rate (IR) of the LSC group was 41.15% +/- 3.86%, 59.24% +/- 5.34% and 69.93% +/- 7.81% at 12, 24 and 48 h, respectively. The IR of the MSC group was 42.82% +/- 4.94%, 62.68% +/- 7.58% and 71.79% +/- 8.12% at 12, 24 and 48 h, respectively. The IR of the HSC group was 37.50% +/- 3.21%, 40.34% +/- 2.98% and 61.99% +/- 4.88% at 12, 24 and 48 h, respectively. These results suggested that a moderate dosage had the most obvious inhibitory efficacy at 48 h. Compared to the DMSO group, the mitosis index of the LSC, MSC, HSC groups was greatly decreased (P < 0.05) at all time points. Any dose of SC-B suppressed mitosis within 12-48 h. Compared to the DMSO group, the percentage of cells in the G0/G1 phase of the MSC group was greatly increased, and that of the S + G2M phase was greatly decreased, while the percentage of cell inhibition (PCI) in the MSC group was greatly increased (P < 0.05). This suggested that SC-B could exclusively arrest cells in the G0/G1 phase. Cyclin D1 mRNA expression was lower in the MSC group than that in the DMSO group (P < 0.05). CONCLUSION: SC-B can inhibit the proliferation and aberrant mitosis of human gastric cancer SCG-7901 cells in vitro. This inhibitory effect may be due to the down-regulation of cyclin D1 mRNA expression, which causes cell cycle arrest of gastric cancer cells.