脂肪酶活力检测方法的比较

对检测脂肪酶活力的实验方法进行了研究。对检测脂肪酶活力的底物选择、底物乳化方法、检测方法等进行了比较。     

Structure Activity Relationships in Alkylammonium C12-Gemini Surfactants Used as Dermal Permeation Enhancers

The purpose of this study was to determine the ability and the safety of a series of alkylammonium C12-gemini surfactants to act as permeation enhancers for three model drugs, namely lidocaine HCl, caffeine, and ketoprofen. In vitro permeation studies across dermatomed porcine skin were performed over 24 h, after pretreating the skin for 1 h with an enhancer solution 0.16 M dissolved in propylene glycol. The highest enhancement ratio (enhancement ratio (ER) = 5.1) was obtained using G12-6-12, resulting in a cumulative amount of permeated lidocaine HCl of 156.5 μg cm⁻². The studies with caffeine and ketoprofen revealed that the most effective gemini surfactant was the one with the shorter spacer, G12-2-12. The use of the latter resulted in an ER of 2.4 and 2.2 in the passive permeation of caffeine and ketoprofen, respectively. However, Azone was found to be the most effective permeation enhancer for ketoprofen, attaining a total of 138.4 μg cm⁻² permeated, 2.7-fold over controls. This work demonstrates that gemini surfactants are effective in terms of increasing the permeation of drugs, especially in the case of hydrophilic ionized compounds, that do not easily cross the stratum corneum. Skin integrity evaluation studies did not indicate the existence of relevant changes in the skin structure after the use of the permeation enhancers, while the cytotoxicity studies allowed establishing a relative cytotoxicity profile including this class of compounds, single chain surfactants, and Azone. A dependence of the toxicity to HEK and to HDF cell lines on the spacer length of the various gemini molecules was found.     

Investigations of Amitraz Neurotoxicity in Rats: Effects on Motor Activity and Inhibition of Monoamine Oxidase

Investigations of Amitraz Neurotoxicity in Rats. III Effectson Motor Activity and Inhibition of Monoamine Oxidase. MOSER,V. C., AND MACPHAIL, R. C. (1989). Fundam. Appl. Toxicol 12,12–22. The formamidine pesticide amitraz (AMZ) producesmany behavioral and physiological changes in rats. We examinedthe dose effect and time course of AMZ on motor activity, monoamineoxidase (MAO) activity, and acetylcholinesterase (AChE) activityto evaluate possible neurochemical mechanisms for the behavioraleffects of AMZ. For motor activity studies, male Long-Evanshooded rats were tested in photocell activity measurement devices.AMZ produced dose-related decreases in motor activity of ratsallowed free access to food and rats maintained at a stablebody weight through food restriction. Lowest effective dosesof AMZ tested were 1–3 mg/kg, administered 20 min beforetesting. AMZ appeared to be about three times more potent infood-restricted rats, indicating that amount of body fat mayplay a significant role in the pharmacokinetics of AMZ. Motoractivity returned to control levels over 4–5 days afterdosing with 100–200 mg/kg AMZ, whereas recovery was evidentthe day after admin istration of low doses (1–30 mg#x002F;kg).Inhibition of MAO was measured in whole brain of rats sacrificedat various times after dosing with AMZ. Only > l00 mg/kgAMZ inhibited MAO, which was measurable within 2 hr of dosingand lasted up to 7 days. AMZ appeared to be more selective fortype B MAO when given in vivo, although MAO-A was also inhibitedat doses 300 mg/kg. However, no selectivity was indicated bythe IC50 values determined in vitro (IC50=31 and 28 µMfor MAO-A and MAO-B, respectively). AMZ produced only negligibleinhibition of AChE at the highest doses administered in vivoor at 10 mM in vitro. These data indicate that while AMZ doesinhibit MAO, the dose range over which it produces this actionis much higher than that which suppressed motor activity. ThusMAO inhibition is probably not responsible for AMZ-induced alterationsin motor activity.     

-514bp及-250bp位点基因多态性对肝脂酶转录活性的影响

目的:探讨同时含肝脂酶(HL)启动子-514 bp和-250 bp多态位点的野生型及变异纯合子型的荧光素酶表达质粒对HL转录活性的影响.方法:PCR法扩增含不同目的基因的DNA片段,将目的基因插入到pUCm-T质粒中,获取含SacⅠ及BglⅡ酶切位点的目的基因,将该基因与含荧光素报告基因的pGL2质粒相连,构建pGL2-HL/-514T+ -250A变异纯合型以及pGL2-HL/-514C+ -250G野生型荧光素酶表达质粒,转染至HepG2细胞内表达,双荧光素酶检测系统检测不同重组质粒荧光素酶报告基因的活性.结果:(1)实验成功构建了同时含HL启动子部位-514/-250位点的野生型和变异纯合型的荧光素酶表达质粒.(2) pGL2-HL/-514T+ -250A变异纯合型的荧光素酶相对表达活性明显低于野生型荧光素酶表达活性(P＜0.01).结论:HL启动子-514及-250位置基因的联合变异可直接影响HL的转录活性.     

The Effects of Zwitterionic Surfactants on Skin Barrier Function

The action of five zwitterionic surfactants on the barrier functionof hairless mouse skin has been studied in vitro. The surfactantsconsidered were dodecylbetaine and hexadecylbetaine (C12BETand C16BET, respectively), hexadecylsul-fobetaine (C16SUB),JV.A'-dimethyl-A'-dodecylamine oxide (C12AO), and dodecyltrimethylam-moniumbromide (C12TAB). Excised skin was pretreated with each surfactant,at various concentrations, for 16 hr, following which the permeationof a model compound, nicotinamide, was measured. The actionof the surfactants was assessed by comparing nicotinamide fluxthrough surfactant-pretreated skin with that across controlmembranes which were exposed to buffer alone for 16 hr. Allsurfactants decreased skin barrier function to some extent Thedegree of nicotinamide penetration enhancement induced was correlatedwith the ratio of the surfactant pretreatment concentrationto the surfactant critical micelle concentration, suggestingthat solubilization of stratum corneum lipids may be an importantmechanism in explaining the effects observed. More detailedstudies with ^l4C-radiolabeled C12BET and C16BET showed thatthe dodecyl analog was itself well absorbed, whereas the C16compound partitioned into the skin favorably but then transferredonly very slowly into the receptor phase. These observationswere consistent with toxicity studies (albeit at much higherconcentrations in a different animal model, the rat) which indicatedthat the dermal LD50 of C12BET was significantly less than thatof C16BET (the value for which was so large that it could notbe reliably determined). Overall, this study provides, we believe,useful information pertinent to the potential dermal toxicityof the surfactants considered following occupational or environmentalexposure, c 1991 Soday of Toxicology.     

表面活性剂及高分子物质对呋喃西林溶解行为及溶液稳定性的影响

目的:探讨不同增溶物质和高分子物质对呋喃西林溶解行为及溶液稳定性的影响。方法:选择表面活性剂如十二烷基硫酸钠（SDS）、吐温80、泊洛沙姆407,及高分子物质如聚维酮K30（PVPK30）、系列羟丙甲基纤维素（HPMC K100、HPMC50、HPMC 30）,配制成系列浓度的溶液,采用紫外分光光度法测定呋喃西林在其中的最大溶解度。同时,考察高温灭菌和低温冷藏后各溶液的稳定性。结果:在考察的表面活性剂或高分子物质浓度0.1～10.0 mg·ml^-1范围内,呋喃西林被增溶的最大饱和溶解度分别为0.245 ng·ml^-1(C_吐温80=5 mg·ml^-1),0.256 mg·ml^-1(C_{泊洛沙坶407}=5 mg·ml^-1),0.385 mg·ml^-1(CSDS=10 mg·ml^-1),0.309 mg·ml^-1(C_PVPK30=10 mg·ml^-1),0.340 mg·ml^-1(C_{HPMC K100}=5 mg·ml^-1),0.401 mg·ml^-1(C_HPMC50=5 mg·ml^-1),0.426 mg·ml^-1(C_{HPMC K30}=10 mg·ml^-1)。在高温灭菌和低温冷藏后稳定性考察中,含有增溶剂的呋喃西林溶液的稳定性显著好于不含增溶剂的呋喃西林溶液。结论:在呋喃西林溶液中加入0.1%～0.5%的HPMC K100更为合理。     

Genotype-Dependent Effects of COMT Inhibition on Cognitive Function in a Highly Specific,Novel Mouse Model of Altered COMT Activity

Catechol-O-methyltransferase (COMT) modulates dopamine levels in the prefrontal cortex. The human gene contains a polymorphism (Val¹⁵⁸Met) that alters enzyme activity and influences PFC function. It has also been linked with cognition and anxiety, but the findings are mixed. We therefore developed a novel mouse model of altered COMT activity. The human Met allele was introduced into the native mouse COMT gene to produce COMT-Met mice, which were compared with their wild-type littermates. The model proved highly specific: COMT-Met mice had reductions in COMT abundance and activity, compared with wild-type mice, explicitly in the absence of off-target changes in the expression of other genes. Despite robust alterations in dopamine metabolism, we found only subtle changes on certain cognitive tasks under baseline conditions (eg, increased spatial novelty preference in COMT-Met mice vs wild-type mice). However, genotype differences emerged after administration of the COMT inhibitor tolcapone: performance of wild-type mice, but not COMT-Met mice, was improved on the 5-choice serial reaction time task after tolcapone administration. There were no changes in anxiety-related behaviors in the tests that we used. Our findings are convergent with human studies of the Val¹⁵⁸Met polymorphism, and suggest that COMT''s effects are most prominent when the dopamine system is challenged. Finally, they demonstrate the importance of considering COMT genotype when examining the therapeutic potential of COMT inhibitors.     

Structure Activity Relationship of Human Microsomal Epoxide Hydrolase Inhibition by Amide and Acid Analogues of Valproic Acid

Purpose. The purpose of this study was to evaluate the in vitro inhibitory potency of various amide analogues and derivatives of valproicacid toward human microsomal epoxide hydrolase (mEH). Methods. mEH inhibition was evaluated in human liver microsomeswith 25 (S)-(+)-styrene oxide as the substrate. Inhibitory potencyexpressed as the median inhibitory concentration (IC₅₀) was calculatedfrom the formation rate of the enzymatic product,(S)-(+)-1-phenyl-1,2-ethanediol. Results. Inhibitory potency was directly correlated with lipophilicityand became significant for amides with a minimum of eight carbonatoms. Branched eight-carbon amides were more potent inhibitors thantheir straight chain isomer, octanamide. N-substituted valproylamideanalogues had reduced or abolished inhibition potency with theexception of valproyl hydroxamic acid being a potent inhibitor. Inhibitionpotency was not stereoselective in two cases of chiral valpromideisomers. Valproyl glycinamide, a new antiepileptic drug currentlyundergoing phase II clinical trials and its major metabolite valproylglycine were weak mEH inhibitors. Acid isomers of valproic acid werenot potent mEH inhibitors. Conclusions. The structural requirements for valproylamide analoguesfor potent in vitro mEH inhibition are: an unsubstituted amide moiety;two saturated alkyl side chains; a minimum of eight carbons in themolecule.     

Kinetic Considerations for the Quantitative Assessment of Efflux Activity and Inhibition: Implications for Understanding and Predicting the Effects of Efflux Inhibition

Purpose Unexpected and complex experimental observations related to efflux transport have been reported in the literature. This work was conducted to develop relationships for efflux activity (PS_efflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), efflux ratio (ER), degree of efflux inhibition (ϕ_i), 50% inhibitory concentration (IC₅₀), and Michaelis–Menten constant (K_m)]. Methods A three-compartment model (apical, cellular, and basolateral) was used to derive flux equations relating the initial rate of flux and steady-state mass transfer in the presence or absence of active efflux. Various definitions of efflux ratio (ER) were examined in terms of permeability-surface area products. The efflux activity (PS_efflux) was expressed in terms of ER and PS. The relationships between PS_efflux and PS, ER, ϕ_i, IC₅₀, and K_m were solved mathematically. Simulations and examples from the literature were used to illustrate the resulting mathematical relationships. Results The relationships derived according to a three-compartment model differed fundamentally from commonly accepted approaches for determining PS_efflux, ϕ_i, IC₅₀ and K_m. Based on the model assumptions and mathematical derivations, currently used mathematical relationships erroneously imply that efflux activity is proportional to change in PS (i.e., flux or P_app) and thus underestimate PS_efflux and ϕ_i, and overestimate IC₅₀ and K_m. Conclusions An understanding of the relationship between efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of efflux transport, and predicting the clinical significance of efflux inhibition.     

鸡卵清蛋白对蛋白酶抑制作用的研究

以新鲜鸡蛋清为材料,采用三氯醋酸-丙酮提取,冷丙酮沉淀,碳酸盐透析和DEAE-Cel-lulose离子交换层析分离出鸡卵清蛋白。研究了该蛋白质对蛋白酶活性的影响,结果表明鸡卵清蛋白对胰蛋白酶有强烈抑制作用并且抑制程度与温度、pH值等密切相关,能部分仰制枯草杆菌蛋白酶活性,不抑制胰凝乳蛋白酶。     

Pokeweed Antiviral Protein,a Ribosome Inactivating Protein: Activity,Inhibition and Prospects

Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics.     

Monoacylglycerol Lipase Inhibition Blocks Chronic Stress-Induced Depressive-Like Behaviors via Activation of mTOR Signaling

The endocannabinoid (eCB) system regulates mood, emotion, and stress coping, and dysregulation of the eCB system is critically involved in pathophysiology of depression. The eCB ligand 2-arachidonoylglycerol (2-AG) is inactivated by monoacylglycerol lipase (MAGL). Using chronic unpredictable mild stress (CUS) as a mouse model of depression, we examined how 2-AG signaling in the hippocampus was altered in depressive-like states and how this alteration contributed to depressive-like behavior. We report that CUS led to impairment of depolarization-induced suppression of inhibition (DSI) in mouse hippocampal CA1 pyramidal neurons, and this deficiency in 2-AG-mediated retrograde synaptic depression was rescued by MAGL inhibitor JZL184. CUS induced depressive-like behaviors and decreased mammalian target of rapamycin (mTOR) activation in the hippocampus, and these biochemical and behavioral abnormalities were ameliorated by chronic JZL184 treatments. The effects of JZL184 were mediated by cannabinoid CB₁ receptors. Genetic deletion of mTOR with adeno-associated viral (AAV) vector carrying the Cre recombinase in the hippocampus of mTORf/f mice recapitulated depressive-like behaviors induced by CUS and abrogated the antidepressant-like effects of chronic JZL184 treatments. Our results suggest that CUS decreases eCB-mTOR signaling in the hippocampus, leading to depressive-like behaviors, whereas MAGL inhibitor JZL184 produces antidepressant-like effects through enhancement of eCB-mTOR signaling.     

Air Pollutant Sulfur Dioxide-induced Alterations on the Levels of Lipids,Lipid Peroxidation and Lipase Activity in Various Regions of the Rat Brain

Abstract: The exposure of rats to SO₂ (10 p.p.m.) for one hour daily for 30 days caused depletion of total lipids in all brain areas. The contents of phospholipid were elevated in cerebellum and brain stem, but were depleted in cerebral hemisphere. Cholesterol levels showed an increase in various brain regions. On the other hand, gangliosides were increased in cerebellum and brain stem, but were decreased in cerebral hemisphere. Interestingly, cholesterol/phospholipid ratio was increased in different regions of the brain. Lipase activity was elevated in cerebral hemisphere. Lipid peroxidation showed marked increment in whole brain and in all the brain areas studied. The results suggest that SO₂-exposure induces degradation of lipids. Interestingly, the lipid contents are affected differentially in the various parts of the brain.     

Cytotoxic Activity and Quantitative Structure Activity Relationships of Arylpropyl Sulfonamides

B13 is a ceramide analogue and apoptosis inducer with potent cytotoxic activity. A series of arylpropyl sulfonamide analogues of B13 were evaluated for their cytotoxicity using MTT assays in prostate cancer PC-3 and leukemia HL-60 cell lines. Some compounds (4, 9, 13, 14, 15, and 20) showed stronger activities than B13 in both tumor cell lines, and compound (15) gave the most potent activity with IC₅₀ values of 29.2 and 20.7 µM, for PC-3and HL-60 cells, respectively. Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis was performed to build highly reliable and predictive CoMSIA models with cross-validated q² values of 0.816 and 0.702, respectively. Our results suggest that long alkyl chains and a 1R, 2R configuration of the propyl group are important for the cytotoxic activities of arylpropyl sulfonamides. Moreover, the introduction of small hydrophobic groups in the phenyl ring and sulfonamide group could increase biological activity.     

Structure Guided Optimization,in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors

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Effects of Antipsychotic Drugs on I to, I Na, I sus, I K1, and hERG: QT Prolongation, Structure Activity Relationship, and Network Analysis

Purpose To evaluate in vitro and computationally model the effects of selected antipsychotic drugs on several ionic currents that contribute to changes in the action potential in cardiac tissue. Methods Fourteen antipsychotic drugs or metabolites were examined to determine whether QT interval prolongation could be accounted for by an effect on one or more myocardial ion channels [I_to, I_Na, I_sus, I_K1, and human ether-a-go-go related gene (hERG)]. Using the patch clamp technique, drug effects on these human cardiac currents were tested. Results All molecules had little inhibitory effect on ion channels (blocking at concentrations >5 μM) other than hERG. A significant correlation was observed between the estimated hERG blockade and the increase in corrected QT for five of the antipsychotics. Molecular modeling identified hydrophobic features related to the interaction with hERG and correctly rank-ordered the test set molecules olanzapine and its metabolites. A network analysis of ligand and protein interactions around hERG using MetaCore™ (GeneGo Inc., St. Joseph, MI, USA) was used to visualize antipsychotics with affinity for this channel and their interactions with other proteins in this database. Conclusion The antipsychotics do not inhibit the ion channels I_to, I_Na, I_sus, I_K1 to any appreciable extent; however, blockade of hERG is a likely mechanism for the prolongation of the QT interval.