Cloning, molecular characterization, and expression pattern of FGF5 in Cashmere goat (Capra hircus).

Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that belongs to the FGF family, and was found to be associated with hair growth in humans and other animals. The Inner Mongolia Cashmere goat (Capra hircus) is a goat breed that provides superior cashmere; this breed was formed by spontaneous mutation in China. Here, we report the cloning, molecular characterization, and expression pattern of the Cashmere goat FGF5. The cloned FGF5 cDNA was 813 base pairs (KM596772), including an open reading frame encoding a 270-amino-acid polypeptide. The nucleotide sequence shared 99% homology with Ovis aries FGF5 (NM_001246263.1). Bioinformatic analysis revealed that FGF5 contained a signal peptide, an FGF domain, and a heparin-binding growth factor/FGF family signature. There was 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 11 protein kinase C phosphorylation sites, 4 casein kinase II phosphorylation sites, 1 amidation site, 1 N-glycosylation site, and 1 tyrosine kinase phosphorylation site in FGF5. Real-time polymerase chain reaction showed that FGF5 mRNA levels were higher in testis than in the pancreas and liver. These data suggest that FGF5 may play a crucial role in Cashmere goat hair growth.

Overexpression of protein kinase B/AKT induces phosphorylation of p70S6K and 4E-BP1 in goat fetal fibroblasts.

Protein kinases regulate many processes, including cell growth, metabolism, molecular interactions, and cell proliferation. Protein kinase B (PKB)/AKT (v-AKT mouse thymoma viral oncogene homolog) is an upstream component of mammalian target of rapamycin (mTOR) signaling and mediates pathophysiological processes in several signaling pathways. This study aimed to construct and overexpress a eukaryotic goat AKT expression vector in goat fetal fibroblasts and examine the effects of AKT on the phosphorylation of p70S6K and 4E-BP1. AKT was subcloned into the expression vector pIRES2-DsRed2 to generate pIRES2-DsRed2-AKT, which was transfected into goat fetal fibroblasts with LipofectamineTM 2000. AKT was measured by reverse transcription-polymerase chain reaction in the transgenic cells, and the expression of AKT and phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) were analyzed by Western blot. Cell clones that stably emitted red fluorescence were obtained after transfection for 48 h, and the exogenous gene was verified. Exogenous AKT was transcribed, and AKT was overexpressed, inducing the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) in goat fetal fibroblasts. Thus, the overexpression of AKT activates mTOR signaling in goat cells.

Prognostic and predictive significance of plasma hepatocyte growth factor and carcinoembryonic antigen in non-small lung cancer after surgery.

OBJECTIVES: Scatter factor, also known as hepatocyte growth factor (SF/HGF), is a polypeptide growth factor with a number of biologic activities, including cell scattering, stimulation of cell motility, mitogenesis, morphogenesis, angiogenesis, and cellular invasiveness, it is thought to be important in the growth and spread of several carcinomas. We assessed whether preoperative plasma levels of HGF and carcinoembryonic antigen (CEA) can enhance the accuracy of standard models for predicting pathologic features and clinical outcomes. PATIENTS AND METHODS: The study comprised 45 consecutive patients treated with surgery for clinically localized non-small-cell lung cancer. HGF and CEA were measured using the commercially available immunoassay. Multivariate logistic regression was used to assess the relationship between plasma HGF/CEA and pathologic features. Multivariate Cox regression was used to predict disease recurrence. RESULTS: Patients with lung squamous cell cancer (SCC) more frequently had higher plasma HGF, whereas CEA levels were significantly elevated in patients with non-SCC histology. Preoperative plasma HGF and CEA levels were not the independent predictors of overall survival. CONCLUSIONS: Preoperative plasma levels of HGF and CEA are not the independent predictors of non-small lung cancer disease recurrence and metastasis after surgery; HGF is a predictor of lung squamous cell cancer. Use of HGF may help in therapeutic decision-making and estimate the histological type of NSCLC.

Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia.

Activation of group I metabotropic glutamate receptors (mGluR) has been implicated in the pathophysiology of acute central nervous system injury. However, the relative roles of the two group I subtypes, mGluR1 or mGluR5, in such injury has not been well examined. We compared the effects of treatment with the newly developed, selective mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) and the selective mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) in a rat intraluminal filament model of temporary middle cerebral artery occlusion (MCAo). Rats were administered MPEP or CHPG i.c.v. beginning 15 or 135 min after induction of ischemia for 2 h. Infarct size was measured after either 22 or 70 h of reperfusion, and neurological function was quantified at 2, 24, 48 and 72 h. Treatment with MPEP or CHPG at 15 min reduced 24 h infarct volume by 61 and 44%, respectively. The neuroprotective effects were dose dependent. Delaying MPEP treatment until 135 min eliminated the neuroprotective effects. In other studies, using early MPEP treatment (15 min) at optimal doses, infarct volume was reduced by 44% at 72 h and this was correlated with significant neurological recovery. These data suggest that both MPEP and CHPG are neuroprotective when administered after focal cerebral ischemia. In separate, recent studies we found that although MPEP does act as an mGluR5 antagonist and blocks agonist induced phosphoinositide hydrolysis, it also serves as a non-competitive NMDA antagonist; in contrast, other results indicate that CHPG mediated neuroprotection may reflect anti-apoptotic activity. Therefore, both types of compounds may prove to have therapeutic potential for the treatment of stroke.

Differential expression of apoptotic protease-activating factor-1 and caspase-3 genes and susceptibility to apoptosis during brain development and after traumatic brain injury.

Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute brain injury. Recent studies have provided evidence that neuronal susceptibility to apoptosis induced by traumatic or ischemic injury decreases during brain development. However, the molecular mechanisms responsible for this age-dependent phenomenon remain unclear. Here we demonstrate that, during brain maturation, the potential of the intrinsic apoptotic pathway is progressively reduced and that such repression is associated with downregulation of apoptotic protease-activating factor-1 (Apaf-1) and caspase-3 gene expression. A similar decline in apoptotic susceptibility associated with downregulation of Apaf-1 expression as a function of developmental age was also found in cultured primary rat cortical neurons. Injury-induced cytochrome c-specific cleavage of caspase-9 followed by activation of caspase-3 in mature brain correlated with marked increases in Apaf-1 and caspase-3 mRNA and protein expression. These results suggest that differential expression of Apaf-1 and caspase-3 genes may underlie regulation of apoptotic susceptibility during brain development, as well as after acute injury to mature brain, through the intrinsic pathway of caspase activation.

Intraventricular vascular endothelial growth factor antibody increases infarct volume following transient cerebral ischemia.

AIM: To clarify the role of vascular endothelial growth factor (VEGF) in neuronal damage induced by cerebral ischemia. METHODS: Expression of VEGF in adult rat brain was measured by immunohistochemistry. Transient middle cerebral artery occlusion (MCAO) model was induced by placing a nylon thread in the lumen of the internal carotid artery. The infarct volume was shown with 2,3,5-triphenyltetrazolium chloride (TTC) staining and quantitated by computer image analyzer with and without VEGF antibody treatment. RESULTS: VEGF expression was widely distributed in neuronal cells besides vascular endothelial cells, and the neuronal distribution of VEGF was specific. After intraventricular treatment with VEGF antibody (0.1 g.L-1 daily, for 7 d following the ischemia), infarct volume in the antibody treatment was increased versus vehicle-treated rats [(21.6 +/- 2.7 vs 16 +/- 6) mm3, P < 0.05] respectively. CONCLUSION: Intraventricular injection of VEGF antibody increased the infarct volume after focal cerebral ischemia in rats, suggesting that expression of neuronal VEGF may be one of neuronal protective mechanisms.

Neuronal ERCC6 mRNA expression in rat brain induced by a transient focal cerebral ischemia.

AIM: To study whether the excision repair cross-complementing group 6 (ERCC6) is involved in the neuronal pathophysiological process following cerebral ischemia-reperfusion injury. METHODS: A transient middle cerebral artery occlusion (MCAO) was used to induce cerebral ischemia-reperfusion injury in rat brain. Northern blot was used to check a specific signal for oligonucleotide probe. The expression of ERCC6 mRNA in the rat brain was observed by in situ hybridization. The specific cellular distribution of ERCC6 mRNA in the neuron or glia of the rat brain was analyzed by double staining combined with confocal laser scanning microscopic analysis. RESULT: The expression of ERCC6 mRNA in the penumbra area increased following ischemia and reperfusion with a time-dependent manner. ERCC6 was expressed on d 2, reached peak values on d 3, and kept high level even on d 14 of reperfusion following ischemia. Number of ERCC6 mRNA expressive cell in the penumbra area on d 1, d 2, d 3, d 7, d 14 of reperfusion following ischemia were (0 +/- 0), (253 +/- 56), (816 +/- 355), (341 +/- 185), (128 +/- 95) x 10(6) cells/m2, respectively. Confocal microscopic analysis showed that ERCC6 mRNA coexpressed with phosphopyruvate hydratase in the neurons and with glial fibrillary acidic protein (GFAP) in a few proliferation astrocyte glia. CONCLUSION: The expression of transcription-repair coupling factor ERCC6 mRNA in the neuron and glia was induced by ischemia-reperfusion injury.

Inhibitory effects of dextromethorphan on c-fos protein expression during focal cerebral ischemia in rats.

AIM: To study the effect of dextromethorphan (DM) in focal cerebral ischemia. METHODS: The c-fos protein was detected immunohistochemically in the brain of rats after focal cerebral ischemia (induced by placing a nylon thread in the lumen of the internal carotid artery) with and without treatment with DM. RESULTS: Focal cerebral ischemia induced c-fos protein expression outside the core territory of the middle cerebral artery (MCA) and neuronal damage in the core territory of the MCA. There was an evident expression of c-fos protein in the ipsilateral regions outside the MCA territory (e.g. cingulate cortices, piriform cortices and entorhinal cortices), and in the contralateral regions of hippocampus after 4-h reperfusion following 1-h MCA occlusion. But morphological results showed severe edema and neuronal damage in the core territory and the ipsilateral hippocampus. DM blocked both the c-fos protein induction and neuronal damage in all regions. CONCLUSION: DM reduced c-fos protein expression and blocked the neuronal damage after focal cerebral ischemia.

Nerve fibers containing dynorphin A in cerebral arteries.

Nerve fibers containing dynorphin (Dyn) A(1-17)-like immunoreactivity were identified around cerebral arteries of guinea pig. The immunoreactive nerve fibers were richly distributed in anterior and middle cerebral arteries, but sparsely in posterior cerebral and basilar arteries. Histofluorescent study showed that large and small cerebral arteries were abundantly innervated by monoamine nerve fibers. Pretreatment with 6-hydroxydopamine or reserpine reduced the concentration of Dyn A in the wall of arteries by about 60% and 30%, respectively. These results demonstrated that there exist Dyn A immunoreactive nerve fibers in cerebral arteries and Dyn A may coexist with monoamine in perivascular nerve fibers.

Effects of sigma and phencyclidine receptor ligands on electric field-stimulated rabbit ear artery constriction in vitro.

Several ligands of phencyclidine (Phe) receptors: Phe, dizocilpine maleate (Diz, MK-801), 1-[1-(2-thionyl)cyclohexyl] piperidine (TCP), and ligands of sigma (sigma) receptors: dl-N-allylnormetazocine (dl-SK&F-10047), 1, 3-di-ortho-tylyl-guanidine (DTG), dl-pentazocine, were tested on rabbit ear arteries in vitro. It was found that the ligands of Phe receptors enhanced the electric field stimulated vasoconstriction (ESV). Their concentration-effect curves of these compounds were parallel in the order of potencies: Phe > Diz > TCP. The ligands of sigma receptors had no effect on ESV of the arteries, but 5 mumol.L-1 reduced or increased the effect of Phe (5 mumol.L-1) on ESV. d-SK&F-10047, d-pentazocine, and DTG inhibited the effect of Phe on ESV from 364 +/- 22 mg to 142 +/- 49 mg (n = 5, P < 0.01), 262 +/- 95 mg (n = 5, P < 0.05), and 291 +/- 80 mg (n = 5, P > 0.05), respectively. The levoisomers: l-SK&F-10047 and l-pentazocine enhanced the effect of Phe on ESV from 364 +/- 22 mg to 484 +/- 78 mg (n = 5, P < 0.05), and 466 +/- 95 mg (n = 5, P < 0.05), respectively. These results revealed that there were mainly Phe receptors but hardly any sigma receptors in the arteries.

Effects of kappa, sigma, and phencyclidine receptors agonists in rat tail arteries of spontaneously hypertensive rats.

The effects of the kappa receptor agonist trans-4-dichloro-N-methyl-N-(2-(1-pyrrolidin)cyclohexyl)-benzen eacefamide methane sulfonate (U-50 488H), etorphine, the sigma (sigma) receptor agonists (+)-3-(3-hydroxychenyl)-N-(1-propyl) piperidine ((+)-3-PPP), 1, 3-di-o-tolyl-guanidine (DTG), and the phencyclidine (Phe) receptor agonists Phe, N-(1-(2-thienyl)cyclohexyl) piperidine (TCP), and dizocilipine maleate (MK-801) on electrically stimulated constriction (ESC) were investigated in the rat tail arteries (RTA) of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Etorphine and U-50 488H inhibited the response to ESC in SHR more than that in WKY. The effects of U-50 488H were greater than those of etorphine. The IC50 and Kact of U-50 488H in SHR were 2.5 +/- 2.0 and 0.43 +/- 0.22 mumol.L-1, respectively, while the corresponding figures in WKY were 23 +/- 15 and 2.3 +/- 1.0 mumol.L-1, respectively (P < 0.05). The inhibitory effects of (+)-3-PPP on ESC in RTA of SHR were weaker than those in WKY. Its IC50 and Kact in SHR were 11.6 +/- 5.4 and 0.87 +/- 0.30 mumol.L-1, respectively, while the corresponding figures in WKY were 0.63 +/- 0.16 and 0.35 +/- 0.18 mumol.L-1, respectively (P < 0.05). But the inhibitory effect of DTG was very slight and the difference of Kact between WKY and SHR was not significant. The enhancing effects of Phe, TCP, and MK-801 in SHR were not at all different from those in WKY at each concentration tested.(ABSTRACT TRUNCATED AT 250 WORDS)

[Antagonistic effect of phencyclidine on cerebral ischemic damage of rats].

Dynorphin and catecholamine were measured in ischemic rat produced by four-vessel (2 vertebral arteries and 2 common carotid arteries) occlusion for 10 min. The results showed that: (1) The contents of dynorphine (pg/mg tissue) in cerebral cortex were 5.5 +/- 0.6 (n = 7) in normal rats and decreased to 4.9 +/- 0.5 (n = 9, P less than 0.05) in cerebral ischemic rats; with immediate ip phencyclidine (1-(1-phenylcyclophexyl)piperidine, PCP, 1 mg.kg-1), the contents of dynorphin were increased to 5.3 +/- 0.4 (n = 5, P less than 0.05 vs the ischemic rats). (2) The contents of DOPAC (pg/mg tissue) in cerebral cortex were 38 +/- 6 (n = 7) and increased to 120 +/- 60 (n = 5, P less than 0.05) in 10 min cerebral ischemic rats; with immediately ip PCP (1 mg.kg-1), the contents of DOPAC were decreased to 26 +/- 13 (n = 7, P less than 0.05 vs the ischemic rats). (3) The release of DA (pg/mg tissue) in cortical slices in vitro, in high K+ solution were 24 +/- 3 (n = 5) and significantly increased to 57 +/- 15 (n = 5, P less than 0.05) in ischemic rat brain slices; with immediate ip PCP (1 mg.kg-1), the contents of DA were decreased to 38 +/- 10 (n = 5, P less than 0.05 vs the ischemic rats). These results suggest PCP play an antagonistic role in cerebral ischemic damage of rats.

[Antagonistic effects of dextromethorphan on vasoconstriction of phencyclidine in vitro].

Bioassay and spectrophotofluorometry were used to study the antagonistic effect of dextromethorphan (DM) on phencyclidine (PCP) vasoconstriction in rabbit ear artery. DM (5 mumols.L-1) antagonized enhancement of PCP, N-[1-(2-thienyl) cyclohexyl] piperidine (TCP) and dizocilpine maleate (MK-801) (5 mumols.L-1) on electrical stimulation-induced vasoconstriction by 86 +/- 18%, 84 +/- 17%, and 86 +/- 18%, respectively (n = 6, P less than 0.01), but had no obvious bioactivity itself at the same concentration. DM (1, 2.5, and 5 mumols.L-1) inhibited the PCP effect and reduced the maximal effect of PCP with pD2' = 5.3 +/- 0.3 (n = 4). The contents of norepinephrine (NE) in control, PCP, and DM + PCP groups were 5 +/- 6, 12 +/- 8, and 5 +/- 6 ng.ml-1, respectively (n = 9). PCP (10 mumols.L-1) increased the NE release (P less than 0.05) but DM (10 mumols.L-1) inhibited it (P less than 0.01). The results suggest DM may be a noncompetitive blockader for PCP receptors.