Recombinant expression in human cells of active integrin α1β1-blocking RTS-disintegrin jerdostatin

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the α₁β₁ integrin. Jerdostatin selectively blocked the adhesion of α₁β₁-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein’s N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble α₁β₁ integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of α₁β₁ integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC₅₀ 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active α₁β₁-blocking jerdostatin in a mammalian cell system.     

Regulation of matrix metalloproteinases (MMP)-2/-9 expression in eosinophilic chronic rhinosinusitis--cell culture by interleukin-5 and -13?

BACKGROUND: Eosinophils are a prominent immunological feature of chronic rhinosinusitis (CRS). Cytokines in the respiratory mucosa may be the key to upper airway pathophysiology. Matrix metalloproteinases (MMP) represent an entire group of Zn2+ dependent endopeptidases with the potential to alter the extracellular matrix (ECM). In this study epithelial cultures of CRS were treated with interleukin (IL)-5 or IL-13 and subsequent levels of metalloproteinases were determined. MATERIALS AND METHODS: The cells for CRS culture were obtained from patients undergoing functional endoscopic sinus surgery. After 8-72 hours incubation with 0.2-0.4 ng/ml IL-5 or 3-6 ng/ml IL-13, the expression of the MMP-2 and -9 in the CRS cultures was analysed. RESULTS: After 72 hours incubation with IL-5, the relative levels of MMP-2 showed no significant alteration in protein expression in comparison with the control groups. Incubation with IL-13 revealed a statistically insignificant decrease of the relative MMP-9 expression in ECRS compared to the control group (p>0.1). CONCLUSION: Alterations of MMP-2 and -9 expression may play a role in ECRS, but the association with IL-5 and IL-13 remains unclear.     

α1-Adrenoceptors in the rat cerebral cortex: New insights into the characterization of α1L- and α1D-adrenoceptors

Among the three α₁-adrenoceptor subtypes (α_1A, α_1B and α_1D) a peculiar intracellular localization and poor coupling to membrane signals of cloned α_1D-adrenoceptor have been reported. In addition, the α_1L-adrenoceptor (low affinity for prazosin), a functional phenotype of α_1A, has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of α₁-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (α_1D> α_1B ? α_1A). α₁-Adrenoceptors were also detected by immunoblotting, revealing α_1A- and α_1B-adrenoceptors to be predominantly expressed in the membrane fraction and the α_1D-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of α_1D-adrenoceptor in tissue homogenates, whereas only α_1A- and α_1B-subtypes were detected in membranes. The proportion of α_1A-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (α_1A/L)-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The α_1A/L-adrenoceptor was heavily implicated in the inositol phosphate response, while the α_1D-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of α_1D-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the α_1L-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the α_1A-adrenoceptor coupled to G-protein.     

Effects of intrathecal 6-hydroxydopamine, α1 and α2 adrenergic receptor antagonists on antinociception of propofol in mice

AIM: To investigate the relationship between spinal cord norepinephrine, alpha1 and alpha2 adrenergic receptors and antinociception of propofol in mice. METHODS: Kunming mice were used. Antinociceptive tests were investigated with the tail-immersion test and the acetic acid-induced writhing test. The effects of subcutaneous (sc), intrathecal (ith) and intracerebroventricular (icv) injection propofol on pain threshold were observed. The influences of pretreatment with ith 6-hydroxydopamine, alpha1R antagonist prazosin, or alpha2R antagonist yohimbine on the antinociception of propofol were studied. RESULTS: Significant antinociception was produced by propofol (25, 50 mg/kg, sc) and propofol (20, 40 microg, ith) in tail-immersion test and acetic the acid-induced writhing test (P<0.05 or P<0.01). Icv propofol (10, 20, and 40 microg) did not produce any effect on pain threshold in mice (P>0.05). The 6-hydroxydopamine (5 and 10 microg), prazosin (5 and 10 microg), or yohimbine (5 and 10 microg) ith alone did not affect basal tail-flick latency (TFL) in conscious mice, but significantly reduced the TFL as measured by tail-immersion test in propofol (50 mg/kg, sc)-treated mice, compared with basal TFL and vehicle groups (P<0.05 or P<0.01). CONCLUSION: The spinal cord is a target of propofol antinociception. In mice propofol antinociception is partly mediated by spinal norepinephrine, alpha1R and alpha2R.     

Effects of morphine and methadone treatment on mRNA expression of Gα(i) subunits in rat brains

Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.     

Effects of captopril and enalaprilat on intracellular Ca~(2 ),Na~ contents and pH in hypoxic and reoxygenated cardiomyocytes

探讨卡托普利（Ｃａｐ）和依那普利拉（Ｅｎａ）保护缺氧和复氧心肌细胞作用机制．方法：采用荧光探针结合图象处理技术测定细胞内离子浓度．结果：缺氧和复氧心肌细胞内Ｃａ２＋（分别为１６５±８和１９６±１４ｎｍｏｌ·Ｌ－１）和Ｎａ＋（分别为９２±０８和９３±１３ｍｍｏｌ·Ｌ－１）高于正常而ｐＨ低于正常（分别为６７±０３和６６１±０１９）．Ｃａｐ和Ｅｎａ减少缺氧细胞内Ｃａ２＋浓度并减轻细胞内酸化．Ｃａｐ也减少缺氧细胞内Ｎａ＋浓度（８１±０９ｍｍｏｌ·Ｌ－１）．Ｃａｐ减少复氧细胞内Ｃａ２＋和Ｎａ＋浓度，但Ｅｎａ无此作用．Ｃａｐ或Ｅｎａ与Ｖｅｒ合用无协同作用．结论：Ｃａｐ和Ｅｎａ对缺氧和复氧心肌细胞有保护作用，但机制并不完全一致．     

Effects of the benzodiazepine GABAA α1-preferring ligand, 3-propoxy-β-carboline hydrochloride (3-PBC), on alcohol seeking and self-administration in baboons

Rationale

The various α subtypes of GABA_A receptors have been strongly implicated in alcohol reinforcement and consumption.

Objectives

The effects of the GABA_A α1-preferring ligand, 3-propoxy-β-carboline hydrochloride (3-PBC), on seeking and self-administration responses were evaluated in two groups of baboons trained under a 3-component chained schedule of reinforcement (CSR).

Methods

Alcohol (4 %?w/v; n?=?5; alcohol group) or a preferred nonalcoholic beverage (n?=?4; control group) was available for self-administration only in component 3 of the CSR. Responses in component 2 provided indices of motivation to drink (seeking). 3-PBC (1.0–30.0 mg/kg) and saline were administered before drinking sessions under both acute and 5-day dosing conditions.

Results

Repeated, and not acute, doses of 3-PBC significantly decreased total self-administration responses (p?<?0.05), volume consumed (p?<?0.05), and gram per kilogram of alcohol (p?<?0.05) in the alcohol group. In the control group, 5-day administration of 3-PBC significantly decreased total self-administration responses (p?<?0.05) but produced nonsignificant decreases in volume consumed. Within-session pattern of drinking was characterized by a high level of drinking in the first 20 min of the session for both groups, which was significantly (p?<?0.05) decreased by all doses of 3-PBC (1.0–18.0 mg/kg) only in the alcohol group. In contrast, the first drinking bout in the control group was only reduced at the highest doses of 3-PBC (10.0 and 18.0 mg/kg).

Conclusions

The results support the involvement of the GABA_A α1 subtype receptor in alcohol reinforcement and consumption.     

Selective potentiation of (α4)3(β2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats

Prefrontal glutamate release evoked through activation of α4β2* nicotinic acetylcholine receptors (nAChRs) situated on thalamic glutamatergic afferents mediates cue detection processes and thus contributes to attentional performance. However, little is known about the respective contributions of the high sensitivity and low sensitivity (LS) stoichiometries of the α4β2 nAChR, (α4)₂(β2)₃ and (α4)₃(β2)₂, to these processes. In the present study we employed glutamate-sensitive microelectrodes and the (α4)₃(β2)₂-selective positive allosteric modulator (PAM) NS9283 to investigate the importance of the LS α4β2 nAChR for glutamate release in the rat medial prefrontal cortex (mPFC). Firstly, the signaling evoked by physiologically relevant ACh concentrations through the (α4)₃(β2)₂ nAChR in HEK293 cells was potentiated by NS9283, consistent with the classification of NS9283 as a PAM. In urethane-anesthetized rats, intra-prefrontal pressure ejections of NS9283 evoked glutamatergic transients. Importantly, this glutamate release was attenuated by removal of cholinergic projections to the recording area. This finding indicates that the effects of NS9283 depend on endogenous ACh, again consistent with effects of a PAM. We then conducted microdialysis to demonstrate the presence of extracellular ACh in urethane-anesthetized control rats. While detectable, those levels were significantly lower than in awake rats. Finally, the amplitudes of glutamatergic transients evoked by local pressure ejections of a low concentration of nicotine were significantly augmented following systemic administration of NS9283 (3.0 mg/kg). In conclusion, our results indicate that a LS α4β2 nAChR PAM such as NS9283 may enhance the cholinergic modulation of glutamatergic neurotransmission in the cortex, thereby perhaps alleviating the attentional impairments common to a range of brain disorders.     

Effects of methyllycaconitine (MLA), an α7 nicotinic receptor antagonist, on nicotine- and cocaine-induced potentiation of brain stimulation reward

It has been shown that nicotine facilitates intracranial self-stimulation (ICSS) reward and that nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) are of primary importance for its reinforcing and dependence-producing actions. Recently, we have shown that α₇ nicotinic receptors in the VTA contribute to both the acute effects of nicotine on the mesolimbic dopamine system, as well as to nicotine withdrawal reactions. However, it is not yet known whether the same receptor conformation is directly involved in the reinforcing actions of nicotine. Here, using the curve-shift method we studied the effects of methyllycaconitine (MLA), a selective α₇ receptor antagonist, microinjected (graded doses: 1, 3, 9 μg/μl per side) into the VTA on the rewarding efficacy of lateral hypothalamic self-stimulation and on the systemic nicotine-induced potentiation of brain stimulation reward. MLA did not affect baseline self-stimulation. Nicotine produced a significant reduction in ICSS threshold, without altering maximal rates of responding, while MLA attenuated the effect of nicotine at the two lower doses. Given the reported interaction between nicotine and cocaine at both the neuronal and the behavioral level, we also examined whether α₇ receptor antagonism within the VTA can affect the reinforcing action of cocaine, as measured with ICSS. Interestingly, MLA attenuated the reinforcing effect of cocaine in all doses tested, without altering the maximal rate of responding, i.e. the performance of the animals. These results suggest that α₇ nAChRs in the VTA are involved in mediating the reinforcing actions of drugs of abuse, such as nicotine and cocaine, and provide evidence that α₇ nAChR antagonists may be clinically useful in attenuating the rewarding effects of addictive drugs. Received: 1 September 1999 / Final version: 18 December 1999     

Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to α₂β₁ integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to α₂β₁ integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant α₂β₁ integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant α₂β₁ integrin nor to cell-surface-exposed α₂β₁ integrin (α₂-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and α₂β₁ integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.     

Effects of α4/β2- and α7-nicotine acetylcholine receptor agonists on prepulse inhibition of the acoustic startle response in rats and mice

RATIONALE: Nicotine and agonists at subtypes of the nicotine acetylcholine receptor (nAChR) affect auditory gating, but the magnitude and direction of such effects appear highly variable. This variability may be due to differences in the tested dose range, selectivity of the test compound, species and strain, and suggests that nAChR subtypes are differentially involved in the control of auditory gating. OBJECTIVES AND METHODS: This study aimed to characterise the effects of nicotine and agonists with preferential activity at alpha4/beta2- and alpha7-nAChRs on auditory sensorimotor gating using a prepulse inhibition (PPI) paradigm. Similar experimental conditions were employed in rats and two strains of mice. The paradigm used startle stimuli of 120 dB and prepulse intensities of 3, 6 and 12 dB above a background of 70 dB. RESULTS: In Sprague-Dawley rats, nicotine disrupted PPI [minimal effective dose (MED): 1 mg/kg, SC] and this effect was mimicked by the potent nAChR agonist, epibatidine, (MED: < or = 0.001 mg/kg, IP) and the potent, and relatively selective, alpha4/beta2-nAChR agonist A-85380 (MED: < or = 0.1 mg/kg, IP). The effects of epibatidine, A-85380 and, to a lesser extent, nicotine were blocked by the non-selective nAChR antagonist mecamylamine. The relatively selective alpha7-nAChR agonists, GTS-21 and AR-R-17779, did not affect PPI in a consistent manner, both in rats and in DBA/2 mice, a strain expressing a disrupted gating phenotype, presumably due to altered activity of hippocampal alpha7-nAChRs. In BALB/c mice, a strain expressing a normal gating phenotype, nicotine (MED: 10 mg/kg, SC), epibatidine (MED: 0.03 mg/kg, IP) and A-85380 (MED: 0.3 mg/kg, IP) predominantly augmented PPI and mecamylamine attenuated these effects. CONCLUSIONS: The present results confirm that the effects of nAChR agonists on PPI are species dependent and suggest that stimulation of heteromeric nAChRs containing both alpha and beta subunits, and possibly of the alpha4/beta2 type, affect sensorimotor gating. Evidence supporting a role for alpha7-nAChRs in the control of PPI of the acoustic startle response was not obtained.     

Effects of α,β-unsaturated sulphones and phosphonium salts on ecto-ATPase activity and contractile responses mediated via P2x-purinoceptors

• 1.1. In the guinea-pig urinary bladder and vas deferens, several α,β-unsaturated sulphones and phosphonium salts that were tested inhibited ecto-ATPase activity. The sulphones were more active in the bladder but the phosphonium salts were more effective in the vas deferens.
• 2.2. These compounds either potentiated or inhibited purinergic contractile responses in the guinea-pig urinary bladder and vas deferens.
• 3.3. α,β-Unsaturated sulphones and phosphonium salts represent a new promising class of compounds, capable of modulating purinergic neurotransmission.

     

Effects of cyclization on stability, structure, and activity of α-conotoxin RgIA at the α9α10 nicotinic acetylcholine receptor and GABA(B) receptor

α-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the α9α10 nicotinic acetylcholine receptor (nAChR) and the GABA(B) receptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3-7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABA(B) receptor over the α9α10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini.     

Effects of isoprenaline,adrenaline and selective α1- and α2- adrenoceptor stimulation on hypoxic pulmonary vasoconstriction in rat isolated perfused lungs

In the rat pulmonary vasculature perfused with blood in situ vasoconstriction induced by hypoxia was reversed by isoprenaline (doses > 1 ng) and adrenaline (doses > 30 ng) and exacerbated by phenylephrine but not UK 14304. Doses of adrenaline < 30 ng had no effect, except in the presence of propranolol (1 μM) or phentolamine (3 μM) when they caused vasoconstriction and vasodilation respectively, showing that, at dose levels < 30 ng, adrenaline's β- adrenoceptor vasodilator properties were balanced by its α- adrenoceptor vasoconstrictor properties. The pressor effects of adrenaline, in the presence of propranolol, were antagonised by prazosin (0.1 μM) but not by equi-molar concentrations of rauwolscine. These results suggest that the α- adrenoceptor agonist property of adrenaline is of benefit to its use as an inhaled bronchodilator because unopposed β- adrenoceptor stimulation can reverse hypoxic pulmonary vasoconstriction in poorly ventilated regions of the lung, promote further ventilation/ perfusion mismatching and lower PaO₂. They further suggest that adrenaline affects pulmonary vascular tone in the rat via α₁- adrenoceptors, stimulation of α₂- adrenoceptors having no effect.     

Effects of dl-3-n-butylphthalide on production of TXB_2 and 6-keto-PGF_(1α) in rat brain during focal cerebral ischemia and reperfusion

目的：观察丁基苯酞（ＮＢＰ）对大鼠局灶性脑缺血及重灌后海马，纹状体和皮层中ＴＸＢ２及６ｋｅｔｏＰＧＦ１α含量的影响．方法：尼龙线栓塞法造成大鼠局灶性脑缺血模型．ＴＸＢ２和６ｋｅｔｏＰＧＦ１α用放免法测定．结果：ＮＢＰ１０ｍｇ·ｋｇ－１治疗对缺血重灌注后脑组织中ＴＸＢ２的产生具有抑制作用，但对６ｋｅｔｏＰＧＦ１α的产生无明显作用．ＮＢＰ２０ｍｇ·ｋｇ－１治疗后，重灌５ｍｉｎ缺血脑组织中ＴＸＢ２和重灌后３０ｍｉｎ时６ｋｅｔｏＰＧＦ１α含量皆明显减少．ＮＢＰ２０或１０ｍｇ·ｋｇ－１皆明显提高ＰＧＩ２／ＴＸＡ２的比值．而阿司匹林（２０ｍｇ·ｋｇ－１）除重灌５ｍｉｎ提高纹状体ＰＧＩ２／ＴＸＡ２的比值外，在其它时间点上均无提高作用．结论：ＮＢＰ提高缺血脑组织中ＰＧＩ２／ＴＸＡ２的比值，可能有利于改善缺血脑组织的微循环状态．     

Decreased carboxylesterases expression and hydrolytic activity in type 2 diabetic mice through Akt/mTOR/HIF-1α/Stra13 pathway

Abstract

• 1.?This study investigated the alteration of carboxylesterases in type 2 diabetes. We found that the carboxylesterase 1d (Ces1d) and carboxylesterase 1e (Ces1e) expression and the capacity of hydrolytic activity of liver and intestine decreased, whereas the Akt/mTOR/HIF-1α/ Stra13 (DEC1) signaling was activated in T2D mice. Consistently, high insulin could give rise to the same results in the high-glucose DMEM condition, which mimicked T2D, in primary mouse hepatocytes.

• 2.?Perifosine or rapamycin almost abolished the decrease of the Ces1d and Ces1e expression and the hydrolytic activity induced by the insulin in the primary mouse hepatocytes.

• 3.?The responsiveness of human hepatoma (HepG2) cells to high insulin in high-glucose condition was similar to that of primary mouse hepatocytes in terms of the altered expression of carboxylesterases.

• 4.?The knockdown of HIF-1α or DEC1 with shRNA construct abrogated the decrease of the CES1 and CES2 expression induced by the insulin in high glucose condition in HepG2 cells.

• 5.?Taken together, the decreased carboxylesterases expression and hydrolytic activity in T2D mice are through the Akt/mTOR/HIF-1α/Stra13 (DEC1) pathway.

     

Effects of tamoxifen, 17α-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female Japanese medaka (Oryzias latipes)

The effects of oral administration of tamoxifen, 17α-ethynylestradiol (EE2), flutamide, and methyltestosterone (MT), on plasma vitellogenin levels of male and female medaka were investigated. Medaka were fed diets containing different concentrations of these chemicals for 7 days, and these plasma vitellogenin levels were measured. Tamoxifen increased significantly the vitellogenin levels in male, but inhibited the normal vitellogenin induction in female in the high concentration groups. EE2 increased significantly vitellogenin levels in both sexes. Flutamide increased significantly the vitellogenin levels in female, but gave no effects on male. MT inhibited the normal vitellogenin induction in female, but increased slightly vitellogenin levels in male without a clear tendency. Administration of tamoxifen, EE2, flutamide, and MT showed the different pattern in vitellogenin levels in both sexes.