Roles of M3 receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Background: This study is to investigate the roles of muscarinic receptor 3 (M₃ receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC).

Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M₃ receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR.

Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M₃ receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M₃ receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M₃ receptor shRNA groups.

Conclusion: These results indicate that M₃ receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M₃ receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M₃ receptor.     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.     

慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin(PV)的表达变化,为其功能的研究提供形态学依据。方法:将30只健康雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组。吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5 mg/kg,逐日递增5mg,至第10d为50mg/kg;对照组注射同体积的生理盐水。于末次注射后动物分别存活3h、3 d和14d。用免疫组化方法和相对平均灰度值检测杏仁核内PV的表达。结果:在生理盐水处理组各存活时间点,杏仁核内PV的表达相同。和生理盐水对照组相比,3h时杏仁核内PV的表达明显增加(P<0.05)。第3d时,杏仁核内PV的表达减少,明显低于第3 h组(P<0.05)。至第14d时,PV的表达又开始增加,明显高于第3 d组(P<0.05)。结论:本结果提示慢性吗啡处理及戒断后杏仁核PV的表达具有时相特异性;这种变化在戒断早期可能主要与躯体依赖相关,而戒断晚期主要与精神依赖相关。     

盐酸多奈哌齐联合综合康复训练对老年痴呆患者认知功能、事件相关电位及血清BDNF、IGF-1的影响

目的:探讨盐酸多奈哌齐联合综合康复训练对老年痴呆患者认知功能、事件相关电位及血清脑源性神经营养因子(BDNF)、胰岛素样生长因子1(IGF-1)的影响。方法:选取2017年3月~2020年3月期间来我院接受治疗的120例老年痴呆患者,根据随机数字表法分为对照组(n=60,盐酸多奈哌齐治疗)和联合组(n=60,盐酸多奈哌齐联合综合康复训练治疗)。对比两组疗效,治疗前、治疗3个月后的认知功能评分、事件相关电位、血清BDNF和IGF-1水平变化,以及治疗期间不良反应发生情况。结果:联合组的临床总有效率为91.67%,高于对照组的70.00%(P<0.05)。两组治疗3个月后日常生活能力评估量表(ADL)、临床痴呆量表(CDR)评分降低,且联合组低于对照组(P<0.05);简易精神状态量表(MMSE)评分升高,且联合组高于对照组(P<0.05)。两组治疗3个月后N1潜伏期、P2潜伏期均缩短,P3波幅均升高,且联合组治疗3个月后P2潜伏期短于对照组,P3波幅高于对照组(P<0.05);两组治疗3个月后N1潜伏期比较未见统计学差异(P>0.05)。联合组治疗3个月后BDNF水平高于对照组(P<0.05);两组治疗3个月后IGF-1水平比较未见统计学差异(P>0.05)。对照组与联合组不良反应发生率对比无统计学差异(P>0.05)。结论:老年痴呆患者在盐酸多奈哌齐基础上联合综合康复训练,认知功能可得到明显改善,同时还可调节患者事件相关电位及血清BDNF、IGF-1水平。     

miR-29a attenuates cardiac hypertrophy through inhibition of PPARδ expression

Although cardiac hypertrophy is widely recognized as a risk factor that leads to cardiac dysfunction and, ultimately, heart failure, the complex mechanisms underlying cardiac hypertrophy remain incompletely characterized. The nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) is involved in the regulation of cardiac lipid metabolism. Here, we describe a novel PPARδ-dependent molecular cascade involving microRNA-29a (miR-29a) and atrial natriuretic factor (ANF), which is reactivated in cardiac hypertrophy. In addition, we identify a novel role of miR-29a, in which it has a cardioprotective function in isoproterenol hydrochloride-induced cardiac hypertrophy by targeting PPARδ and downregulating ANF. Finally, we provide evidence that miR-29a reduces the isoproterenol hydrochloride-induced cardiac hypertrophy response, thereby underlining the potential clinical relevance of miR-29a in which it may serve as a potent therapeutic target for heart hypertrophy treatment.     

A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding

The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liquid chromatography, and size-exclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half-lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively, at 25 degrees C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT-3 the slowest to refold and BDNF the fastest. Threading of the N-terminus out through the cystine-knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine-knot loop (14 for hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.     

Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode     

Electroacupuncture Facilitates Recovery of Male Sexual Behavior in Morphine Withdrawal Rats

The effect of electroacupuncture (EA) on the sexual behavior of male rats undergoing morphine withdrawal was studied by measuring various parameters of sexual behavior. In addition, the total serum testosterone (TST) concentrations in male rats at different times of morphine administration and abstinence were measured. Acute and chronic administration of morphine severely inhibited the sexual behavior of the rats and lowered their TST concentrations. TST concentrations recovered to normal within 24 h after the last morphine injection, while sexual behavior remained suppressed for at least 7 days. Electroacupuncture (2/100 Hz alternately) administered once daily for 7 days during morphine withdrawal facilitated the recovery of male sexual behavior and increased TST concentrations to above normal. The effect of EA on sexual behavior may involve both neuronal and hormonal pathways.     

Thermal and guanidine hydrochloride-induced denaturation of human cystatin C

Wild-type human cystatin C is directly involved in pathological fibrils formation, leading to hemorrhage, dementia and eventually death of people suffering from cerebral amyloid angiopathy. Some studies on cystatin C oligomerization have been already done but some points are still unclear. In order to learn more about this important process, we have investigated thermal and chemical (guanidine hydrochloride-induced) denaturation of human cystatin C. Studies performed using tryptophan fluorescence, calorimetry, circular dichroism and Fourier transform infrared spectroscopy demonstrate that neither chemical nor thermal denaturation of hCC are simple two-state events. One recognized intermediate form was dimeric cystatin C, whose appearance was preceded mainly by changes in the L2 binding loop. The other form occurred only in the chemical denaturation process and was characterized by partially recovered interactions maintaining the protein tertiary structure. Our studies also strongly indicate that the -structural motif of cystatin C is directly implicated in formation of temperature-induced aggregates.Abbreviations Gdn.HCl guanidine hydrochloride - hCC human cystatin C     

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.