Functional status of microvascular vasomotion is impaired in spontaneously hypertensive rat.

Accumulating evidence demonstrates that microcirculation plays a role in the pathogenesis of hypertension. In the current study, we demonstrated that pancreatic islet microvascular vasomotion of spontaneously hypertensive rats (SHRs) lost the ability to regulate blood flow perfusion and exhibited a lower microvascular blood perfusion pattern which was negative correlated with blood glucose level. SHRs administrated with insulin revealed an improvement of pancreatic islet microvascular vasomotion and blood perfusion pattern. In vitro, the expressions of endothelial nitric oxide synthase (eNOS) and phospho-eNOSser1177 (p-eNOSser1177) were significantly decreased in high glucose exposed islet endothelial cells (iECs), accompanied with a higher ratio of eNOS monomer to eNOS dimer and a significantly increased malondialdehyde and nitrite levels. Meanwhile, barrier function, tube formation and migration capacities of high glucose exposed iECs were significantly inhibited. In contrast, iECs dysfunction induced by glucose toxicity and oxidative stress was attenuated or improved by supplement with insulin, L-arginine and ß-mercaptoethanol. In summary, our findings suggest that functional status of pancreatic islet microvascular vasomotion is impaired in SHRs and provide evidence that treatment with insulin, L-arginine and ß-mercaptoethanol improves endothelium-dependent microvascular vasomotion and meliorates iECs function due to anti-hyperglycemic and anti-oxidative effects, partly through mechanism involving regulation of eNOS and p-eNOSser1177.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on ß-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture.

This study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 µM cysteamine (C+C); 100 IU catalase (CAT) or 100 µM ß-mercaptoethanol (ß-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66-100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6-48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6-93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.

Effects of thiol antioxidant ß-mercaptoethanol on diet-induced obese mice.

AIMS: Obesity and insulin resistance are associated with increased oxidant stress. However, treatments of obese subjects with different types of antioxidants often give mixed outcomes. In this work, we sought to determine if long-term supplementation of a thiol antioxidant, ß-mercaptoethanol, to diet-induced obese mice may improve their health conditions. MAIN METHODS: Middle-age mice with pre-existing diet-induced obesity were provided with low concentration ß-mercaptoethanol (BME) in drinking water for six months. Animals were assessed for body composition, gripping strength, spontaneous physical and metabolic activities, as well as insulin and pyruvate tolerance tests. Markers of inflammation were assessed in plasma, fat tissue, and liver. KEY FINDINGS: BME-treated mice gained less fat mass and more lean mass than the control animals. They also showed increased nocturnal locomotion and respiration, as well as greater gripping strength. BME reduced plasma lipid peroxidation, decreased abdominal fat tissue inflammation, reduced fat infiltration into muscle and liver, and reduced liver and plasma C-reactive protein. However, BME was found to desensitize insulin signaling in vivo, an effect also confirmed by in vitro experiments. SIGNIFICANCE: Long-term supplementation of low dose thiol antioxidant BME improved functional outcomes in animals with pre-existing obesity. Additional studies are needed to address the treatment impact on insulin sensitivity if a therapeutic value is to be explored.

Comparative effects of adding ß-mercaptoethanol or L-ascorbic acid to culture or vitrification-warming media on IVF porcine embryos.

The aims of the present study were to; (1) determine the effects of supplementation with two antioxidants during in vitro culture (IVC) on embryo development and quality; and (2) test the effects of adding the antioxidants to vitrification-warming media on the cryotolerance of in vitro-produced (IVP) porcine blastocysts. In Experiment 1, presumptive zygotes were cultured without antioxidants, with 50 µM ß-mercaptoethanol (ß-ME) or with 100 µM L-ascorbic acid (AC). After culture, blastocyst yield, quality and cryotolerance were evaluated in each treatment group. In Experiment 2, survival rates (3 and 24 h), total cell number, apoptosis index and the formation of reactive oxygen species (ROS) in blastocysts vitrified-warmed with 100 µM AC or 50 µM ß-ME or without antioxidants added to the vitrification medium were compared. Antioxidant addition during IVC had no effect on embryo development, total cell number or the apoptosis index, and culturing embryos in the presence of ß-ME had no effects on cryotolerance. In contrast, ROS levels and survival rates after vitrification-warming were significantly improved in embryos cultured with AC. Furthermore, addition of AC into vitrification-warming media enhanced embryo survival and embryo quality after warming. In conclusion, our results suggest that supplementing culture or vitrification media with 100 µM AC improves the quality and cryosurvival of IVP porcine blastocysts.

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav.

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27 kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121 kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata' proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.

Thiols stabilize cobblestone morphology of cultured mesothelial cells.

Cellular thiols including GSH (glutathione) and L-Cys (L-cysteine) are essential for cell signalling, growth and differentiation. L-Cys is derived from the extracellular thiol pool and is the rate-limiting compound for intracellular GSH biosynthesis. The present study investigated the effect of thiol-supplemented medium on cell growth, phenotype and total GSH of cultured hPMCs (human peritoneal mesothelial cells). Cells were cultured in medium M199 supplemented with 2% serum, with 'plus' or without 'minus' L-Cys and compared with medium supplemented with either ß-ME (ß-mercaptoethanol) (0.25 mmol/l) or the receptor tyrosine kinase ligand EGF (epidermal growth factor, 100 ng/ml). ß-ME produced a disproportionate increase in total GSH compared with L-Cys and other thiols tested [(procysteine (2-oxothiazolidine-4-carboxylic acid) or NAC (N-acetyl-L-cysteine)], while growth and morphology were identical. Cell behaviour of primary hPMCs is characterized by the transition of fibroblastoid to cobblestone morphology during early passage. L-Cys and ß-ME promoted a rapid MET (mesenchymal-to-epithelial transition) within 3 days of culture, confirmed by the presence of cobblestone cells, intact organelles, abundant microvilli, primary cilia and cortical actin. In contrast, EGF produced a biphasic response consisting of delayed growth and retention of a fibroblastoid morphology. During a rapid log phase of growth, MET was accompanied by rapid catch-up growth. Thiols may stabilize the epithelial phenotype by engaging redox-sensitive receptors and transcription factors that modulate differentiation. These data may benefit researchers working on thiol-mediated cell differentiation and strategies to regenerate damage to serosal membranes.

Gating of aquaporins by heavy metals in Allium cepa L. epidermal cells.

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn(2+), Pb(2+), Cd(2+), Hg(2+)). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 µM-2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that ß-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20-40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating.

Synergistic effects of glutathione and ß-mercaptoethanol treatment during in vitro maturation of porcine oocytes on early embryonic development in a culture system supplemented with L-cysteine.

Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O(2)) tension and the addition of antioxidants. The beneficial effects of antioxidants and O(2) tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), ß-mercaptoethanol (ß-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or ß-ME (25 µM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 µM ß-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or ß -ME. These GSH- and ß-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or ß-ME with L-cysteine significantly reduced high O(2) tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 µM ß-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems.

The effects of various antioxidants on the development of parthenogenetic porcine embryos.

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, ß-mercaptoethanol (ß-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 µM ß-ME; 17.2% and 17.5% for 50 and 100 µM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, ß-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P < 0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P < 0.05). When the reactive oxygen species levels in two-cell embryos after 1 µM ß-ME and 100 µM α-tocopherol treatment were examined, those were lower than control group (P < 0.05). In conclusion, it was found that the three antioxidants, ß-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.

A potential antimicrobial treatment against ESBL-producing Klebsiella pneumoniae using the tellurium compound AS101.

Due to the extensive spread of antibiotic-resistant Klebsiella pneumoniae, the non-toxic immunomodulator, ammonium trichloro (dioxoethylene-o, o') tellurate (AS101), was introduced for the first time in this study. Eleven strains of K. pneumoniae were tested: five were extended spectrum beta lactamase (ESBL)-producing strains and six were non-ESBL-producing strains. The MIC and MBC of ten strains were 9 microg/ml AS101 and 18 microg/ml for one strain. AS101 treatment inhibited bacterial growth in a dose-dependent manner on protein-rich media. No inhibition by AS101 was observed on poorer media. In combination with beta-mercaptoethanol (2-ME) or cysteamine, AS101 inhibited bacterial growth in both types of media. Growth inhibition was also shown following AS101 treatment at both lag and log phases. Our data indicate that AS101 enters the bacterium through its porins, causing bacterial destruction. The mechanism of cell death was characterized using several techniques: (a) scanning electron microscopy showed that bacteria treated with AS101 or in combination with cysteamine exhibited evidence of cell-wall damage; (b) X-ray microanalysis demonstrated damage to Na/K pumps; and (c) transmission electron microscopy demonstrated cell lysis. These phenomena suggest that AS101 has antibacterial potential against K. pneumoniae infections.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.

Effect of 2-mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormone-stimulated lambs.

The objective was to determine the effect of 2-mercaptoethanol and cysteine on in vitro developmental competence of oocytes from lambs (4-8-week old) stimulated with eCG and pFSH. Oocytes were matured in medium (TCM199) with no supplement (Control group) or with 100 microM 2-mercaptoethanol and 600 microM cysteine (GSH group). Oocytes from adult sheep were also included (Adult group). The addition of 2-mercaptoethanol and cysteine did not improve nuclear maturation or microtubule configuration 12, 15, 18, or 24 h after placement in maturation medium. Sperm head decondensation and male pronucleus formation were evaluated at 6, 12, and 18 h after commencement of IVF; sperm decondensation appeared earlier in the GSH group (6h after the start of IVF). There were differences (P<0.05) between the Control group and the GSH and Adult groups for: fertilization rate at both 12 h (55.4, 77.0, and 80.6%, respectively) and 18 h (67.9, 86.9, and 88.7%); parthenogenesis rate at both 12 h (25.0, 10.8, and 5.6%) and 18 h (28.3, 9.8, and 4.5%); and polyspermy rate at 18 h (26.4, 4.9, and 5.7%). Blastocyst rate at 7d was higher in the GSH group than the Control group (23.9% vs. 14.9%, P<0.05), but both were lower (P<0.05) than the Adult group (38.3%). The addition of 2-mercaptoethanol and cysteine improved sperm decondensation and rates of fertilization and the blastocyst development to 7d, with no effect on blastocyst rate at 9 d.

Bovine in vitro oocyte maturation as a model for manipulation of the gamma-glutamyl cycle and intraoocyte glutathione.

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br.

This work evaluated the in vitro cytotoxic activity of laticifer proteins (LP) recovered from the latex of the medicinal plant Calotropis procera. The LP displayed considerable cytotoxicity with IC(50) values ranging from 0.42 to 1.36 microg/ml to SF295 and MDA-MB-435 cell lines, respectively. In healthy peripheral blood mononuclear cells exposed to LP (10 microg/ml) for 72 h, no noticeable effects on viability or cell morphology were seen. The fractionating of LP on an ion exchange chromatography gave rise to a new fraction (PI) that retained almost all cytotoxicity. The cytotoxic effects of both LP and PI were diminished when previously treated with pronase, or 2-mercaptoethanol, suggesting a protein nature of active molecules, however, pre-incubation with dithiothreitol (DTT) only reduced PI activity. PI did not exhibit cysteine proteinase activity, indicating that cysteine proteinases, abundantly found in LP, are not implicated in LP cytotoxicity. In this study, using HL-60 cell as a model, LP was shown to inhibit DNA synthesis. This is probably due to alterations in the topology of DNA, since it was observed that LP is able to interfere in topoisomerase I activity by somehow acting upon DNA. LP provoked reduction in cell number but it did not cause any significant increase in the number of non-viable cells. These findings corroborated with the morphologic analysis, where cells treated with LP showed morphology of apoptotic process with abundant vacuoles, chromatin condensation and fragmentation of the nuclei. The results of this study suggests that LP is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines.

Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cells can terminally differentiate into all types of somatic cells and are considered a promising source of seed cells for tissue engineering. However, despite recent progress in in vitro differentiation and in vivo transplantation methodologies of ES cells, to date, no one has succeeded in using ES cells in tissue engineering for generation of somatic tissues in vitro for potential transplantation therapy. METHODS AND RESULTS: ES-D3 cells were cultured in a slow-turning lateral vessel for mass production of embryoid bodies. The embryoid bodies were then induced to differentiate into cardiomyocytes in a medium supplemented with 1% ascorbic acid. The ES cell-derived cardiomyocytes were then enriched by Percoll gradient centrifugation. The enriched cardiomyocytes were mixed with liquid type I collagen supplemented with Matrigel to construct engineered cardiac tissue (ECT). After in vitro stretching for 7 days, the ECT can beat synchronously and respond to physical and pharmaceutical stimulation. Histological, immunohistochemical, and transmission electron microscopic studies further indicate that the ECTs both structurally and functionally resemble neonatal native cardiac muscle. Markers related to undifferentiated ES cell contamination were not found in reverse transcriptase-polymerase chain reaction analysis of the Percoll-enriched cardiomyocytes. No teratoma formation was observed in the ECTs implanted subcutaneously in nude mice for 4 weeks. CONCLUSIONS: ES cells can be used as a source of seed cells for cardiac tissue engineering. Additional work remains to demonstrate engraftment of the engineered heart tissue in the case of cardiac defects and its functional integrity within the host's remaining healthy cardiac tissue.

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro.

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.

Determination of selectivity and efficacy of fatty acid synthesis inhibitors.

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

The effect of levamisole combined with the classical treatment in chronic brucellosis.

Levamisole is an immunopotenciator drug which is used as an antihelmintic drug as well as very effective remedy on cellular immunity compared with humoral immunity. A total 71 patients (37 men, 34 women) who referred to our department between March 1997 and December 2001, with a history of the disease for about 1 year, were diagnosed as having chronic brucellosis through those tests brucella serum agglutination test (SAT), SAT with Coombs and SAT with 2-mercaptoethanol. The patients were randomly divided into levamisole group (36 patients) and control group (35 patients). All patients were given rifampicin 600 mg/day + doxycycline 200 mg/day for 6 weeks as a standard classical combined therapy for brucellosis. In the levamisole group, oral levamisole 80 mg every other day for 6 weeks was added to the treatment. There was a statistically significant difference between two groups, in complaints of arthralgia, fatigue and sweats before and 6 months after treatment, as well as in erythrocyte sedimentation rate and C-reactive protein elevations and lymphomonocytosis finding. While it was provided both clinical and serological improvement in all patients in the levamisole group; 11 patients in the control group did not improve both clinically and in view of specific and nonspecific laboratory findings and a recurrence occurred in one case, in this group. In conclusion, levamisole added to classical antibiotic therapy in treatment of chronic brucellosis was found quite efficient in all patients in providing adequate clinical and laboratory response in comparison to classical antibiotic therapy alone.