mU mL-1 Research Articles

Biological Effects of Red Chili Pepper (Capsicum annuum) Consumption on High Fat Diet Female Albino Rats.

The evidence for safe consumption of chili pepper to gain health benefits is insufficient as high doses reported to cause gastric erosion. The objective of this study was to investigate the contribution of red chili pepper ethanol extract (RCEE) to weight loss in high fat dieted female albino Wistar rats while monitoring adverse effects of high dose consumption. Body weight gain, lipid profile, oxidative stress parameters, liver, kidney and thyroid biochemical functions were determined in addition to histopathological examination of liver and kidney to determine biological effects of 2 ingested concentrations of red chili pepper extract (5 and 10 mg). Red chili pepper ethanol extract (RCEE) showed anti-obesity potentials and hypolipidemic effects through decreased levels of total cholesterol (T.Ch), low density lipoprotein (LDL), triglycerides (TG) and very low density lipoprotein (VLDL) reached 25, 55, 45 and 45%, respectively, in parallel with increased high density lipoprotein (HDL) levels by 22.6%. The antioxidative potentials of RCEE were pronounced through significant increases in glutathione peroxidase (GPx) and catalase (CAT) activities (346.43 mU mL-1 and 15.33 U L-1 in group 2, respectively). Histological examination of liver and kidney showed adverse results but that did not significantly reflect on their functional biochemical parameters. Results introduced scientific evidence that red chili pepper possesses functional pharmacological properties but the excessive consumption induces liver and kidney damage.

A ratiometric fluorescent biosensor for the sensitive determination of α-glucosidase activity and acarbose based on N-doped carbon dots.

In this work, a novel ratiometric fluorescent platform for α-glucosidase (α-glu) and its inhibitor was constructed based on N-doped carbon dots (N-CDs). The α-glucosidase present can catalyze the release of hydroquinone (HQ) from α-arbutin. Then, the generated HQ can be oxidized and copolymerized with polyethyleneimine (PEI) to form a yellowish green fluorescence copolymer (PHQ-PEI) with intense fluorescence emission at 510 nm. When the PHQ-PEI was formed, blue fluorescence of N-CDs at 425 nm was decreased, whereas the fluorescence of PHQ-PEI at 510 nm increased sharply as a result of the fluorescence resonance energy transfer (FRET) effect between N-CDs and PHQ-PEI. However, in the presence of acarbose, the activity of α-glucosidase is inhibited, and α-arbutin cannot be hydrolyzed to hydroquinone, leading to the fluorescence recovery of N-CDs at 425 nm and the fluorescence decrease of PHQ-PEI at 510 nm. The linear range from 0.2 to 1.6 mU mL-1 and 25-150 μmol L-1 was obtained for α-glucosidase and acarbose detection, respectively, and the detection limit (LOD) for α-glucosidase and acarbose was as low as 0.082 mU mL-1 and 14.5 μmol L-1. Thus, a ratiometric fluorescent sensor with good sensitivity and high specificity was established for α-glucosidase assay and satisfactory results were acquired in real sample determination.

Ratiometric sensors with selective fluorescence enhancement effects based on photonic crystals for the determination of acetylcholinesterase and its inhibitor.

Ratiometric fluorescent sensors are powerful tools for quantitative analyses. However, gold nano-clusters (AuNCs) as typical fluorophores in ratiometric sensors have some disadvantages, such as low luminous efficiency. In this study, a highly sensitive ratiometric fluorescence sensor was fabricated by the combination of AuNCs and fluorescein (FL), and the photonic crystals (PhCs) were used to selectively enhance the fluorescence intensity of AuNCs. This fluorescence sensor was used for the sensitive detection of acetylcholinesterase (AChE) and its inhibitor paraoxon. AChE can catalyze the hydrolysis of acetylthiocholine (ATCh) to form thiocholine (TCh), which can induce the fluorescence quenching of AuNCs while having no obvious influence on the fluorescence intensity of FL. AChE can be determined in the range from 0.1 to 25 mU mL-1 with a limit of detection (LOD) of 0.027 mU mL-1, and paraoxon can be determined in the range of 0.06 to 60 ng mL-1 with a LOD 0.025 ng mL-1. This method, as a new way to selectively improve the fluorescence signal of one of the fluorophores in the ratiometric sensor, would be a promising strategy for the sensitive determination of AChE and its inhibitor.

Gold nanoclusters-based dual-channel assay for colorimetric and turn-on fluorescent sensing of alkaline phosphatase

Here, based on their bifunctional peroxidase-mimicking activity and fluorescence of bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs), we develop a colorimetric and fluorescent dual-channel assay for alkaline phosphatase activity determination. The peroxidase-like activity of BSA-AuNCs makes it possible to catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into its blue product (oxTMB) in the presence of H2O2, and result in the fluorescence quench of BSA-AuNCs simultaneously due to the inner filter effect between oxTMB and BSA-AuNCs. While the ascorbic acid generating form alkaline phosphatase (ALP)-catalyzed hydrolysis of L-ascorbic acid-2-phosphate can inhibit the oxidation of TMB, thereby inducing decolorization of oxTMB and fluorescence recovery of BSA-AuNCs. Based on these findings, a dual-readout assay for ALP activity sensing using BSA-AuNCs has been reported for the first time. The detection limitation can reach as low as 0.26 and 0.16 mU mL-1 by colorimetric and fluorescent method, respectively. Moreover, the developed assay has been successfully applied to detect ALP in human serum samples with satisfactory results. This method may not only offer new idea for simple, sensitive and accurate detection of ALP activity, but also broaden the applications of BSA-AuNCs in bioanalysis.

Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity

Phosphorylation of proteins catalyzed by protein kinases (PKs) is essential to many biological processes; the sensitive detection of PK activity and the screening of PK inhibitors are thus integral to disease diagnosis and drug discovery. Herein, a highly sensitive biosensor has been fabricated for the electrochemical detection of PK activity by exploiting the electrochemically controlled reversible addition-fragmentation chain transfer (eRAFT) polymerization as a novel amplification strategy. The fabrication of the eRAFT-polymerization-based electrochemical biosensor involves (1) the immobilization of substrate peptides onto a gold electrode by way of gold-sulfur self-assembly, (2) the site-specific phosphorylation of substrate peptides by PKs, (3) the anchoring of carboxyl-group-containing chain transfer agents (CTAs) to the phosphorylated sites, and (4) the eRAFT polymerization under a potentiostatic condition, using ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through the eRAFT polymerization, long polymer chains containing numerous electroactive Fc tags can be de novo grafted from each phosphorylated site, resulting in significant amplification of the electrochemical detection signal. The as-fabricated biosensor is highly selective and features a very low detection limit of 1.02 mU mL-1, in the presence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent PK (PKA) as the model target. Results also demonstrate that it can be applied to the screening of PK inhibitors and the detection of PK activity in complex serum samples and cell lysates. Moreover, it holds the merits of easy fabrication, high efficiency, and low cost, which make it a promising tool for the detection of PK activity and the screening of potential PK inhibitors.

Peroxidase-like activity of acetylcholine-based colorimetric detection of acetylcholinesterase activity and an organophosphorus inhibitor.

Colorimetric detection of acetylcholinesterase (AChE) and its inhibitor organophosphates (OPs) is attractive for its convenience, but the addition of exogenous catalyst to produce a chromogenic agent may result in complexity and interference. Herein, we first found that acetylcholine (ATCh) itself mimicked peroxidase's activity, based on which a simple and reliable colorimetric system containing ATCh- 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 was developed for the sensitive and selective assay of AChE activity and its inhibitor OPs. Due to the AChE-catalyzed hydrolysis of acetylcholine, the peroxidase-like activity was affected, which was used for highly sensitive detection of AChE activity with a low limit of detection (LOD) of 0.5 mU mL-1 and a linear detection range from 2.0 to 14 mU mL-1. Furthermore, due to the inhibition of OPs on AChE, OPs were also detected with the present ATCh regulated colorimetric system with LOD of 4.0 ng mL-1 and a linear dynamic range from 10 to 10 000 µg L-1. This strategy was also demonstrated to be applicable for pesticide detection in real samples. Meanwhile, the sensing platform can also be implemented on test strips for rapid and visual monitoring of OPs. Thus, this extremely simple colorimetric strategy without the addition of other exogenous catalysts holds great promise for on-site pesticide detection and can be further exploited for sensing applications in the environmental and food safety fields.

Metal coordination polymer induced perylene probe excimer fluorescence and its application in acetylcholinesterase sensing and alpha-fetoprotein immunoassay.

A novel sensing strategy for acetylcholinesterase (AChE) and alpha-fetoprotein (AFP) is developed, based on the perylene probe monomer to excimer fluorescence transformation induced by the in situ generation of a metal coordination polymer. In the presence of AChE, acetylthiocholine chloride was hydrolyzed to thiocholine. Ag+ and the produced thiocholine formed a positively charged metal coordination polymer, which induced the aggregation of a negatively charged perylene probe and the formation of probe excimer emission. The intensity ratio of excimer to monomer emission was proportional to the AChE concentration. A sensing method for AChE was thus established with a detection limit of 0.02 mU mL-1. The excimer emission with a large Stokes shift could avoid the interference of background fluorescence from complex biological samples, and thus achieved selective and sensitive detection of AChE. In addition, a fluorescence immunoassay strategy for AFP was then developed. Gold nanoparticles (AuNPs) co-immobilized with acetylcholinesterase and the AFP antibody as the capture and amplification probe were first prepared. In the presence of AFP, the sandwich structure was formed by immunological recognition. The hydrolysis of acetylthiocholine was catalyzed by AChE on the AuNPs, and the metal coordination polymer was then formed which resulted in the aggregation of the perylene probe and the formation of the excimer emission. The proposed sensing method offers a new strategy for the detection of other biomarkers.

Metal-to-Ligand Charge-Transfer-based Visual Detection of Alkaline Phosphatase Activity

The ability to directly detect alkaline phosphatase (ALP) activity in undiluted serum samples is of great importance for clinical diagnosis. In this work, we report the use of the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of the Cu(BCA)2+ (BCA = bicinchoninic acid) reporter for the visual detection of ALP activity. In the presence of ALP, the substrate ascorbic acid 2-phosphate (AAP) can be enzymatically hydrolyzed to release ascorbic acid (AA), which in turn reduces Cu2+ to Cu+. Subsequently, the complexation of Cu+ with the BCA ligand generates the chromogenic Cu(BCA)2+ reporter, accompanied by a color change of colorless-to-purple of the solution with a sharp absorption band at 562 nm. The underlying MLCT-based mechanism has been demonstrated on the basis of density functional theory (DFT) calculations. Needless of any sequential multistep operations and elaborately designed colorimetric probe, the proposed MLCT-based method allows for a fast and sensitive visual detection of ALP activity within a broad linear range of 20 - 200 mU mL-1 (R2 = 0.999), with a detection limit of 1.25 mU mL-1. The results also indicate that it is highly selective and has great potential for the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum samples. Owing to the prominent simplicity and practicability, it is reasonable to conclude that the proposed MLCT-based method has a high application prospect in clinical diagnosis.

Fluorescence assay for alkaline phosphatase based on ATP hydrolysis-triggered dissociation of cerium coordination polymer nanoparticles.

Alkaline phosphatase (ALP) is a significant biomarker for diagnostics. Simple, selective and sensitive detection of ALP activity is thus of critical importance. In this study, an artful fluorescence assay for ALP is proposed based on adenosine triphosphate (ATP) hydrolysis-triggered disassociation and fluorescence quenching of cerium coordination polymer nanoparticles (CPNs). ATP, a recognized natural substrate of phosphatase, can serve as a superb "antenna" to sensitize the luminescence of Ce3+ with the aid of tris(hydroxymethyl) aminomethane (Tris), forming Ce3+-ATP-Tris CPNs. In the presence of ALP, ATP will be catalytically converted into adenosine and inorganic orthophosphate, however neither of them can sensitize Ce3+ in alkaline media. As a result, the obtained CPNs are disassociated, inducing the quenching of the fluorescence. On this basis, a straightforward fluorescence assay for ALP activity is rationally developed. The fluorescence quenching efficiency shows a linear relationship for ALP within the activity range from 0.1 to 10 mU mL-1 with a detection limit of 0.09 mU mL-1 under the optimal experimental conditions. Moreover, this facile yet effective fluorescence method featured simplicity, cost-effectiveness, high sensitivity and high selectivity and can be successfully utilized for the quantitative detection of ALP in human serum samples.

Synthesis and Functional Characterization of Caffeic Acid Glucoside Using Leuconostoc mesenteroides Dextransucrase.

Caffeic acid was modified via transglucosylation using sucrose and dextransucrase from Leuconostoc mesenteroides B-512FMCM. Following enzymatic modification, a caffeic acid glucoside was isolated by butanol separation, silica gel chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a molecular mass-to-charge ratio of 365 m/z, and its structure was identified as caffeic acid-3-O-α-d-glucopyranoside. The production of this caffeic acid-3-O-α-d-glucopyranoside at a concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and 650 mU mL-1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the caffeic acid-3-O-α-d-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold higher antilipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth, and 11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-d-glucopyranoside may be a suitable functional component of food and pharmaceutical products.

Oxidase-mimicking activity of ultrathin MnO2 nanosheets in colorimetric assay of acetylcholinesterase activity.

In the present study, a novel colorimetric sensing platform was constructed for quantitative detection of acetylcholinesterase (AChE) activity and its inhibitor. Manganese dioxide (MnO2) nanosheets as an oxidase-mimicking nanomaterial could directly oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxTMB without the need for horseradish peroxidase and H2O2. When AChE was introduced, acetylthiocholine could be catalytically hydrolyzed to produce thiocholine, which easily triggers the decomposition of MnO2 nanosheets, causing the decrease of solution absorbance. Owing to the inhibition effect of organophosphorus pesticides, the enzymatic activity was suppressed, preventing the decomposition of MnO2 and resulting in the increase of absorbance. Under optimal conditions, the colorimetric platform shows sensitive responses to AChE and paraoxon in the range of 0.1-15 mU mL-1 and 0.001-0.1 μg mL-1, respectively. The detection limits of AChE and paraoxon reached 35 μU mL-1 and 1.0 ng mL-1, respectively. Furthermore, the MnO2-TMB platform has been used to fabricate test strips for rapid and convenient visual detection of AChE and its inhibitor with highly promising performance.

Turn‐On Colorimetric Platform for Dual Activity Detection of Acid and Alkaline Phosphatase in Human Whole Blood

The activity detection of acid phosphatase (ACP) and alkaline phosphatase (ALP) is of great importance to the diagnosis and prognosis of related diseases. In this work, we report for the first time a turn-on colorimetric platform for the activity detection of ACP and ALP, by exploiting Cu(BCDS)22- (BCDS=bathocuproinedisulfonate) as the probe. The presence of ACP or ALP dephosphorylates the substrate ascorbic acid 2-phosphate to produce ascorbic acid, which then reduces Cu(BCDS)22- into Cu(BCDS)23- , leading to a turn-on spectral absorption at 484 nm and a dramatic color change of the solution from colorless to orange-red. The underlying metal-to-ligand charge-transfer mechanism has been demonstrated by quantum mechanical computations. This platform allows a rapid, sensitive readout of ACP and ALP activities within the dynamic range from 0 to 220 mU ml-1 . In addition, it is highly immune to false-positive results and also highly selective. More importantly, it is applicable in the presence of human serum and even whole blood samples. These results demonstrate that our platform holds great potential in clinical practices and in the point-of-care analysis.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Immunity to Pneumococcal Vaccine in Splenectomized β-Thalassemia Patients

Problem statement: Splenectomy is accompanied by a lifelong risk of overwhelming post splenectomy infection, mainly caused by encapsulated bacteria such as Streptococcus pneumoniae. The mortality rate in those infected patients remains high. Therefore the pneumococcal polysaccharide vaccine has been recommended. Approach: To obtain information on the immunity levels of pneumococcal antibody, in splenectomized β-thalassemia patients in south of Iran. Patients and methods: This descriptive and cross sectional study was carried out on 263 splenectomized β-thalassemia patients in south of Iran, June 2007-September 2008. Anti-pneumococcal vaccine antibody concentrations were measured by the enzyme-linked immunosorbent assay method. The patients were divided into two groups based on antibody concentrations: Group 1: Hyporesponsiveness patients or non-immune with less than 250 mU mL-1 antibody concentrations and group 2: Immune patients or responder with equal or more than 250 mU mL-1 antibody concentrations. Results: The results showed that 33.80 and 66.20% of the patients were immune and non-immune to pneumococcal infections, respectively. There was an inverse significant between Immunity level with the period after pneumococcal vaccination (r = -0.683, p = 0.0001). Conclusion: These results suggest that a high percentage splenectomized β-thalassemia patients are poor responders to pneumococcal vaccination. Therefore evaluations immunity levels of pneumococcal vaccine and re-vaccination for pneumococcus are recommended in these patients.

80 USE OF THE SHORT DISPOSABLE NEEDLE SYSTEM FOR FOLLICLE ASPIRATION IN MARES

To achieve success in equine somatic cell nuclear transfer (SCNT), it is important to obtain recipient oocytes of good quality. Transvaginal ultrasoundguided follicle aspiration (TVUFA) is one of the methods to obtain recipient oocytes in equine SCNT, but the commercial long double-lumen needle for TVUFA in large animals is not currently purchasable. The aims of the present study were (1) to compare the recovery rate of short disposable needle system (14G) with that of the long double-lumen needle (12G) and (2) to investigate the developmental competency of recovered oocytes after SCNT and embryo transfer (ET). A real-time ultrasound scanner (Mylab30 vet, Esaote, Italy) equipped with a 7.5-MHz convex array transducer (model EC123) housed in a plastic vaginal device with stainless steel needle guidance was used for TVUFA from pre-ovulatory follicles 25 to 40 mm in diameter on synchronized thoroughbred mares between 6 and 9 years of age. Two types of needles were used: (1) a 12G double-lumen long needle (V-EOAD-1260L; Cook, Brisbane, Australia); (2) a 14G single-lumen disposable needle (2.1 × 80mm; Bovi-vet, Kruuse, Denmark) inserted with 18G inner needle (0.8 × 600 mm) using stainless steel connector and tube. Recovered oocytes were matured in vitro in TCM-199 with 5 mU mL-1 FSH (Folltropin-V, Bioniche, Belleville, ON, Canada) and 10% fetal bovine serum (Sigma, St. Louis, MO, USA) for 12 to 16 h. Matured oocytes were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm-1, 20μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 1 mM 6-DMAP. Immediately following SCNT procedures, cloned embryos were surgically transferred to the oviducts of recipient mares (n = 2 to 5 embryos per recipient) that had ovulated within 24 hours before the transfer. An initial pregnancy examination was performed using transrectal ultrasonography between Days 14 and 16 (Day 0 = surgery). The recovery rate of the short disposable needle (n = 89, 44.1%) was slightly increased compared with that of the long needle (n = 34, 29.8%), but the difference was not significant. Nineteen SCNT embryos were transferred to 8 mares, and 1 mare is maintaining pregnancy for 60 days. The results of this study demonstrated that our short disposable needle system could be used instead of the commercial long needle and in vivo development of oocyte recovered with the 14G needle could be maintained after nuclear transfer and embryo transfer.

High Quality Film Nanoimprint Technology for Blu-Ray Disc (BD) Media

Thermal nanoimprint of roll to roll technology for DVD (t=0.6 mm) media realized fast 8 discs/s process and a new platen film nanoimprint process with stamper cooling method resolved the film shrinkage issue. The speed is as fast as 1 s and the film thickness is 75 mum. Dual layer Blu-Ray based disk of injection mold (t=1.1 mm) L0 layer and nanoimprinted (t=75 mum) L1 layer shows signal jitter around 6.0% high quality characteristics

317 ANTI-HYALURONIDASE ACTION OF ELLAGIC ACID EFFECTIVELY PREVENTS POLYSPERMY THROUGH SUPPRESSION OF THE ACROSOME REACTION INDUCED BY THE SPERM - ZONA INTERACTION DURING PORCINE IN VITRO FERTILIZATION

We previously reported that the anti-hyaluronidase agents oligosaccharide and tannic acid (TA) were efficient probes for promoting the normal fertilization process in terms of an effective decrease in the incidence of polyspermy, not only in cumulus-enclosed but also in denuded oocytes in pigs. It was unclear, however, why the polyspermic penetration into the zona pellucida (ZP) was directly prevented by the anti-hyaluronidase action. The present study was conducted to examine the effects of 3 tannin relatives [TA, gallic acid (GA), and ellagic acid (EA)] on IVF parameters and the acrosome reaction induced by the sperm–ZP interaction. The anti-hyaluronidase and radical-scavenging activities of tannin relatives were measured by the colorimetric and the DPPH methods, respectively. Porcine cumulus–oocyte complexes (COCs) were cultured for 44 h in 0.1 mL of TCM-199 supplemented with 0.6 mM cysteine, 40 mU mL-1 of FSH, 20 mU mL-1 of LH, and 10% porcine follicular fluid. After in vitro maturation (IVM), the COCs were freed from their cumulus cells and inseminated by frozen-thawed ejaculated sperm in modified Tris-buffered medium (IVF medium) containing 0 (control) or 5 �g mL-1 of tannin relatives. After 2 h of co-incubation, the oocytes were gently pipetted to remove loosely bound sperm and stained with Hoechst 33342 to count the number of ZP-bound sperm, or stained with fluorescein isothiocyanate (FITC)-PNA, PI, and 422,6-diamidino-2-phenylindole to evaluate the acrosomal status. At 10 h post-insemination, IVF parameters were examined by lacmoid staining. The data were analyzed by ANOVA and the Tukey-Kramer test. None of the tannin relatives caused a protective proteolytic modification of the ZP matrix or a reduction of the acrosomal proteolytic activity or the number of ZP-bound sperm. There was no difference in the sperm penetration rate even in the presence of tannin relatives (73-82%). However, the incidence of polyspermy was remarkably prevented by TA (32%; 31/98) and EA (21%; 20/94) compared with the control (58%; 58/100; P < 0.05), resulting from their strong anti-hyaluronidase actions, whereas GA without the anti-hyaluronidase action had no effect on the prevention of polyspermy (51%; 43/84). The rate of acrosome reaction induced by the sperm–ZP interaction was decreased by TA (15%; 123/833) and EA (16%; 110/708) compared with the control (25%; 238/939; P < 0.05), and a similar result was found in sperm binding to the pretreated ZP with 500 U of hyaluronidase for 2 h (18%; 351/1959). Interestingly, when sperm were incubated in IVF medium with 10 �g mL-1 of progesterone for 0.5 h to induce a compulsory acrosome reaction instead of the ZP, EA never disturbed the acrosome reaction (23%; 98/424) as control (23%; 102/437), although this reaction was blocked by TA (13%; 57/427) and GA (13%; 50/375), which possessed higher levels of radical-scavenging activity than EA (P < 0.05). These results indicate that the anti-hyaluronidase action of TA and EA effectively prevented polyspermy during porcine IVF as a consequence of suppression of the acrosome reaction functionally induced by the sperm–ZP interaction requiring the hyaluronidase intervention.

Effects of electrical stimulation and insulin on Na+-K+-ATPase ([3H]ouabain binding) in rat skeletal muscle.

Exercise has been reported to increase the Na+-K+-ATPase (Na+-K+ pump) alpha2 isoform in the plasma membrane 1.2- to 1.9-fold, purportedly reflecting Na+-K+ pump translocation from an undefined intracellular pool. We examined whether Na+-K+ pump stimulation, elicited by muscle contraction or insulin, increases the plasma membrane Na+-K+ pump content ([3H]ouabain binding) in muscles from young rats. Stimulation of isolated soleus muscle for 10 s at 120 Hz caused a rapid rise in intracellular Na+ content, followed by an 18-fold increase in the Na+ re-extrusion rate (80 % of theoretical maximum). Muscles frozen immediately or 120 s after 10-120 s stimulation showed 10-22 % decrease in [3H]ouabain binding expressed per gram wet weight, but with no significant change expressed per gram dry weight. In soleus muscles from adult rats, [3H]ouabain binding was unaltered after 10 s stimulation at 120 Hz. Extensor digitorum longus (EDL) muscles stimulated for 10-60 s at 120 Hz showed no significant change in [3H]ouabain binding. Insulin (100 mU ml-1) decreased intracellular Na+ content by 27 % and increased 86Rb uptake by 23 % soleus muscles, but [3H]ouabain binding was unchanged. After stimulation for 30 s at 60 Hz soleus muscle showed a 30% decrease in intracellular Na+ content, demonstrating increased Na+-K+ pump activity, but [3H]ouabain binding measured 5 to 120 min after stimulation was unchanged. Stimulation of soleus or EDL muscles for 120-240 min at 1 Hz (continuously) or 10 Hz (intermittently) produced no change in [3H]ouabain binding per gram dry weight. In conclusion, the stimulating effects of electrical stimulation or insulin on active Na+, K+-transport in rat skeletal muscle could not be even partially accounted for by an acute increase in the content of functional Na+ -K+ pumps in the plasma membrane.

Bradykinin is not involved in angiotensin converting enzyme modulation of ovarian steroidogenesis and prostaglandin production in frog Rana esculenta

Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17beta-estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse-phase high-pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 microM), bradykinin receptor antagonist NPC 567 (1 mg mL-1), bradykinin fragment (1-7) (10 microM), ACE (2.5 mU mL-1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17beta-estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE-modulated pathway.

Effect of acute exogenous hyperinsulinaemia on very low density lipoprotein subfraction composition in normal subjects.

Subtle abnormalities of very-low-density lipoprotein (VLDL) composition and distribution seem to be associated with increased cardiovascular risk. The aims of this study were first, to evaluate whether hyperinsulinaemia per se is able to produce VLDL abnormalities and second, whether this occurs through a stimulation of lipolytic enzymes. Eight normal male volunteers, age 36 +/- 7 years (M +/- SD), body mass index (BMI) 26+/-3 kg m-2, underwent a 5-h euglycaemic hyperinsulinaemic clamp (1.2 mU insulin/kg b.w. min-1). Nine sex, age and BMI comparable subjects underwent control experiments (saline infusion). Three VLDL subfractions of decreasing size were isolated by density gradient ultracentrifugation; lipoprotein lipase (LPL) and hepatic lipase (HL) post-heparin plasma activities were determined by the 3H-labelled triolein method. Hyperinsulinaemia ( approximately 65 mU mL-1) produced the expected plasma free fatty acid suppression. Triglyceride levels were reduced in total VLDL (- 27 +/- 32% vs. + 38 +/- 52% after saline, P < 0.05) and in the larger VLDL (- 56 +/- 19 vs. + 34 +/- 38, P < 0.001). Moreover the relative contribution of the larger subfraction was decreased (- 39 +/- 15% vs. - 3 +/- 21%, P < 0.01), while the percentage of smaller particles was increased (+17 +/- 20 vs. - 9 +/- 22, P < 0.05). LPL and HL activities were decreased to the same degree during either insulin or saline infusion. Exogenous hyperinsulinaemia produced lipoprotein abnormalities partially similar to those previously shown in type 1 diabetic patients, indicating that these abnormalities may be secondary to insulin therapy.