An investigation of the antibacterial effect of carrot on Listeria monocytogenes

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10 000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Effects of low concentrations of cetyltrimethylammonium bromide on diluted bovine spermatozoa

Cetyltrimethylammonium bromide (CTAB) at a concentration of 10 mg/l in a citrate buffer partially protected sperm against the effects of cold shock treatment. CTAB concentrations of 10 and 20 mg/l also increased in vitro sperm livability when semen was diluted in Caprogen and incubated at 37 degrees C (50 h v. 60 and 57 h). Concentrations of CTAB exceeding 50 mg/l reduced in vitro sperm livability. The addition of catalase (4.5 mg/l) to Caprogen produced greater increases in in vitro sperm livability (50 h v. 110 h), but did not eliminate the toxic effect of the higher CTAB concentrations. The addition of CTAB (10 mg/l) to the Caprogen-catalase diluent significantly increased the non-return rate of the diluted semen in only one of four large-scale field trials and did not alter non-return rates with deep-frozen semen.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Oxalate modulates thiobarbituric acid reactive species (TBARS) production in supernatants of homogenates from rat brain, liver and kidney: effect of diphenyl diselenide and diphenyl ditelluride

The aim of this paper was to investigate the mechanism(s) involved in the sodium oxalate pro-oxidative activity in vitro and the potential protection by diphenyl diselenide ((PhSe)(2)) and diphenyl ditelluride ((PhTe)(2)) using supernatants of homogenates from brain, liver and kidney. Oxalate causes a significant increase in the TBARS (thiobarbituric acid reactive species) production up to 4mmol/l and it had antioxidant activity from 8 to 16mmol/l in the brain and liver. Oxalate had no effect in kidney homogenates. The difference among tissues may be related to the formation of insoluble crystal of oxalate in kidney, but not in liver and brain homogenates. (PhSe)(2) and (PhTe)(2) reduced both basal and oxalate-induced TBARS in rat brain homogenates, whereas in liver homogenates they were antioxidant only on oxalate-induced TBARS production. (PhSe)(2) showed a modest effect on renal TBARS production, whereas (PhTe)(2) did not modulate TBARS in kidney preparations. Oxalate at 2mmol/l did not change deoxyribose degradation induced by Fe(2+) plus H(2)O(2), whereas at 20mmol/l it significantly prevents its degradation. Oxalate (up to 4mmol/l) did not alter iron (10micromol/l)-induced TBARS production in the brain preparations, whereas at 8mmol/l onwards it prevents iron effect. In liver preparations, oxalate amplifies iron pro-oxidant activity up to 4mmol/l, preventing iron-induced TBARS production at 16mmol/l onwards. These results support the antioxidant effect of organochalcogens against oxalate-induced TBARS production. In addition, our results suggest that oxalate pro- and antioxidant activity in vitro could be related to its interactions with iron ions.     

Stability of firefly luciferase in Tricine buffer and in a commercial enzyme stabilizer

A solution of firefly luciferase in AuthentiZyme Enzyme Stabilizer® retains full activity when stored in an ice bath (0.5°C) during one day. These solutions have the advantage that no additional protein (other than the luciferase) is present, which is desirable for proteolytic digestion and protein dervatization experiments. For longer-term experiments, firefly luciferase solutions in 0.05 mol/l Tricine buffer at pH 7.8, 10 mmol/l MgSO₄, 1 mmol/l EDTA, and 1 mmol/I DTT which contain 100μg/ml of bovine serum albumin are stable for 6 weeks if frozen and thawed only once.     

Purification of 120-Kilodalton Phytochrome from Oryza Sativa L

The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiolated rice (Oryza sativa L. subsp, japonica var. nongken 58) seedlings. Approximately l kg of 6d old seedlings (the first 2 days at 33℃, the last 4 days at 27 ℃ in darkness) were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer, at final pH 8.45 (4 ℃). After polyethylenimine precipitation, phytochrome in extract was converted to Pfr by irradiation of the resulting supernatant for 10 min with red light. The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ratio of 10 ml per phytochrome unit. The pellet precipitated with ammonium sulfate at 42% saturation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8 ml instead of 0.65 ml per phytochrome unit. Then it was washed successively with 200 mmol/l and 100 mmol/1 phosphate buffer (0.85 ml per phytochrome unit). Native phytochrome (120 kDa) in 12% yield was dissolved in 2 mmol/l EHPES buffer (2.2 ml per phytochrome unit, pH 7.8, containing 5 mmol/l EDTA and 14 mmol/l 2-mercaptoethanol) was proved to be pure in SDS- polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome.     

Insulin resistance in offspring of hypertensive parents.

OBJECTIVE--To determine if insulin resistance is present in normotensive adults at increased risk of developing hypertension. DESIGN--Normotensive subjects with at least one hypertensive parent were paired with offspring of normotensive parents (controls), being matched for age, sex, social class, and physical activity. SETTING--Outpatient clinic. SUBJECTS--30 paired subjects (16 men and 14 women) with and without a family history of hypertension, aged 18-32, with a body mass index < 25 kg/m2, with blood pressure < 130/85 mm Hg, and not taking drugs. INTERVENTIONS--Euglycaemic glucose clamp (two hour infusion of insulin 1 mU/kg/min) and intravenous glucose tolerance test (injection of 100 ml 20% glucose). MAIN OUTCOME MEASURES--Insulin mediated glucose disposal and insulin secretion. RESULTS--The offspring of hypertensive parents had slightly higher blood pressure than did the controls (mean 117 (SD 6) v 108 (5) mm Hg systolic, p = 0.013; 76 (7) v 67 (6) mm Hg diastolic, p = 0.017). Their insulin mediated glucose disposal was lower than that of controls (29.5 (6.5) v 40.1 (8.6) mumol/kg/min, p = 0.002), but, after adjustment for blood pressure, the difference was not significant (difference 6.9 (95% confidence interval -1.5 to 15.3), p = 0.10). Insulin secretion in the first hour after injection of glucose was slightly but not significantly higher in the offspring of hypertensive patients (9320 (5484) v 6723 (3751) pmol.min/l). The two groups had similar concentrations of plasma glucose (5.2 (0.3) v 5.1 (0.4) mmol/l), serum cholesterol (4.4 (0.8) v 4.6 (0.8) mmol/l), serum triglyceride (0.89 (0.52) v 0.68 (0.27) mmol/l), and serum low density lipoprotein cholesterol (2.81 (0.65) v 2.79 (0.61) mmol/l). The offspring of hypertensive parents, however, had lower serum concentrations of high density lipoprotein cholesterol (1.24 (0.31) v 1.56 (0.35) mmol/l, p = 0.002) and higher serum concentrations of non-esterified fatty acids (0.7 (0.4) v 0.4 (0.4) mmol/l, p = 0.039). CONCLUSIONS--Young normotensive subjects who are at increased risk of developing hypertension are insulin resistant.     

Salt and the glycaemic response.

The possibility that salt increases plasma glucose and insulin responses to starchy foods was investigated. Six healthy adults took four morning test meals randomly: 50 g carbohydrate as cooked lentils or white bread, with or without 4.25 g of added salt (an amount within the range of salt found in a meal). When salt was added to the lentils the incremental area under the three hour plasma glucose curve was significantly greater than that for lentils alone (43.2 mmol.min/l v 11.1 mmol.min/l (778 mg.min/100 ml v 200 mg.min/100 ml]. When salt was added to bread the peak glucose concentration was significantly higher than that for unsalted bread (6.96 mmol/l v 6.35 mmol/l (125 mg/100 ml v 114 mg/100 ml], and this was followed by relative hypoglycaemia. Plasma insulin concentrations at 45 minutes were higher after a meal of salted lentils and salted bread than after the unsalted foods (p less than 0.05). The high insulin concentration after salted bread was sustained for one hour after the meal, thus the mean area under the three hour curve was 39% greater than that for unsalted bread (p less than 0.05). Salt may increase the postprandial plasma glucose and insulin responses to lentils and bread by accelerating the digestion of starch by stimulating amylase activity or accelerating small intestinal absorption of the liberated glucose, or both. The findings of this preliminary study, if confirmed by others, would support the recommendation that diabetics, as well as the general population, should reduce their intake of salt.     

The lethal effect of carrot on Listeria species

When shredded or sliced carrots were inoculated with Listeria monocytogenes the number of viable listerias decreased rapidly. On carrot slices stored at 8 degrees C there was a decrease after 3d followed by an increase, after 7d, to numbers similar to those present initially. The numbers of spoilage micro-organisms increased throughout storage at 8 degrees C. Carrots macerated in a Stomacher Lab Blender also showed an antilisterial activity which resulted in a decrease in number of viable bacteria and in sublethal damage. The effect was shown by five carrot cultivars and acted on nine strains of L. monocytogenes and single strains of L. innocua, L. ivanovii, L. seeligeri, L. melshimeri. This antilisterial effect was heat-labile, was inactivated after a few hours at 4 degrees C or at 30 degrees C and was active over the pH range 5.8 to 7.0. Maceration of carrots in an Atomix blender for 1 min or in liquid nitrogen destroyed the antilisterial activity.     

The lethal effect of carrot on Listeria species

When shredded or sliced carrots were inoculated with Listeria monocytogenes the number of viable listerias decreased rapidly. On carrot slices stored at 8°C there was a decrease after 3 d followed by an increase, after 7 d, to numbers similar to those present initially. The numbers of spoilage micro-organisms increased throughout storage at 8°C. Carrots macerated in a Stomacher Lab Blender also showed an antilisterial activity which resulted in a decrease in number of viable bacteria and in sublethal damage. The effect was shown by five carrot cultivars and acted on nine strains of L. monocytogenes and single strains of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri . This antilisterial effect was heat-labile, was inactivated after a few hours at 4°C or at 30°C and was active over the pH range 5.8 to 7.0. Maceration of carrots in an Atomix blender for 1 min or in liquid nitrogen destroyed the antilisterial activity.     

Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia,advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine

Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to long-term complications of diabetes such as impaired wound-healing and retinopathy.We report the effect of increased glucose concentrations, glycated basic fibroblast growth factor (FGF-2) and bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) on the proliferation of bovine aortic endothelial cells.Glucose (30 and 50 mmol/l) had an antiproliferative effect on endothelial cells. This effect may be mediated through reduced mitogenic activity of FGF-2. The glycation of FGF-2 with 250 mmol/l glucose-6-phosphate led to reduced mitogenic activity compared to native FGF-2. BSA-AGE at concentrations of 10, 50 and 250 g/ml had an antiproliferative effect on cultured endothelial cells.Aminosalicylic acid at a concentration of 200 mol/l proved to be more effective than equimolar concentrations of aminoguanidine in protecting endothelial cells against the antiproliferative effects of both high (30 mmol/l) glucose and 50 g/ml BSA-AGE. FGF-2 glycated in the presence of 4 mmol/l aminosalicylic acid or aminoguanidine retained mitogenic activity compared to that glycated in their absence.Compounds like aminoguanidine and, in particular, aminosalicylic acid protect endothelial cells against glucose-mediated toxicity and may therefore have therapeutic potential.     

Effect of supranutritional level of dietary alpha-tocopheryl acetate and selenium on rabbit semen

This research examined the effects of dietary alpha-tocopheryl acetate (50 or 200 mg/kg diet) and selenium (Se, 0 or 0.5 ppm) supplementation on motion characteristics, oxidative stability and fertilizing ability of rabbit spermatozoa, fresh and stored for 24 h at 5 degrees C. The higher amount of dietary alpha-tocopheryl acetate increased the level of Vitamin E in the fresh semen (1.75 mmol/l versus 0.95 mmol/l) and its oxidative stability (thiobarbituric acid reactive substances-TBARS 12.44 nmol malondialdehyde/10(8) sperm versus 21.4 nmol malondialdehyde/10(8) sperm). Dietary Se increased gluthatione peroxidase activity (GPx) in erythrocytes (285 U/g Hb versus 207 U/g Hb), seminal plasma (270 U/l versus 190 U/l) and spermatozoa (1338 mU/10(9) sperm versus 1103 mU/10(9) sperm), whereas it did not show any effect on alpha-tocopherol level and TBARS. No synergy between Vitamin E and Se was shown. Storage for 24 h at 5 degrees C increased the TBARS level in all the experimental groups. Neither live and acrosome reacted spermatozoa, nor kinetic parameters, nor fertility rate were modified by dietary supplementation.     

Subcutaneous continuous insulin infusion and control of blood glucose concentration in diabetics in third trimester of pregnancy.

The effect of subcutaneous continuous insulin infusion on the control of blood glucose concentrations was assessed in eight pregnant diabetics in the third trimester. Twenty-four-hour glucose profiles were obtained after strict inpatient control on conventional insulin regimens and after the start of the continuous infusion, which was maintained for 5-55 days. Mean 24-hour glucose concentrations (6.2 mmol/l on conventional regimen, 5.9 mmol/l on continuous infusion; 111.6 and 106.2 mg/100 ml respectively) and mean fasting concentrations (5.3 v 6.2 mmol/l; 95.4 v 111.6 mg/100 ml) were not significantly changed by continuous infusion. Diurnal variations in glucose concentration tended to be smaller on continuous infusion: standard deviation from mean 24-hour glucose concentration was reduced from 2.5 to 2.0 mmol/l (from 45 to 36 mg/100 ml), maximum excursion from 8.4 to 7.4 mmol/l (151.2 to 133.2 mg/100 ml), and M value from 16 to 14. Subcutaneous continuous insulin infusion may be useful in limiting diurnal variations of blood glucose concentrations and warrants further investigation since such an action may be beneficial in the management of pregnant diabetics, in whom the best possible control of blood glucose concentrations is sought for the good of the fetus.     

9-Aminoacridine- and tetraethylammonium-induced reduction of the potassium permeability in pancreatic B-cells. Effects on insulin release and electrical properties.

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05--1 mmol/l) and tetraethylammonium (2--40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb+ efflux from the islets. Both drugs also slightly reduced 86Rb+ uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.     

Adenosine induced direct negative inotropic effect is abolished during global ischemia: role of protein kinase C and prostacyclin

Adenosine acts as a cardioprotective agent by producing coronary vasodilation, decreasing heart rate and by antagonizing the cardiostimulatory effect of catecholamines; adenosine also exerts a direct negative inotropic effect. Myocardial ischemia is known to be associated with enhanced levels of adenosine, increased protein kinase C (PKC) activity and prostacyclin (PGI2) release. The present study was conducted to determine if myocardial ischemia alters the cardioprotective effect of adenosine by increasing PKC activity and PGI2 release in the isolated rat heart perfused at 10 ml/min with Krebs-Henseleit buffer (KHB; 95% O2+5% CO2). Adenosine (10 mmol/min) reduced myocardial contractility as indicated by a decrease in contractility (dp/dtmax), heart rate (HR) and coronary perfusion pressure (PP). In hearts subjected to 30 min of ischemia (without perfusion) and then reperfused with KHB, adenosine failed to decrease dp/dtmax, HR or PP. However, during infusion of PKC inhibitor H-7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine hydrochloride) (H-7; 6 mmol/min), which commenced 10 min before ischemia and continued throughout reperfusion, adenosine produced a decrease in dp/dtmax, HR and PP, similar to that before ischemia. Infusion of the PKC activator phorbol 12,13-dibutyrate (PDBu; 2 nmol/min) but not an inactive analogue in non-ischemic hearts prevented the adenosine induced decrease in dp/dtmax. During infusion of H-7, PDBu failed to block the direct negative inotropic effect of adenosine in non-ischemic hearts. In addition, pretreatment with H-7 or indomethacin (cyclooxygenase inhibitor) significantly reduced the PGI2 release following ischemia. This data suggest that PKC and PGI2 regulate the direct negative inotropic effect of adenosine, which is abolished during ischemia.     

Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P < 0.05), with the exception of embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P < 0.05). The experimental group did not affect the total number of cells per blastocyst. In conclusion, this study showed that supplementation of the CM with EGF and IGF could partially avoid the deleterious effect of in vitro culture of small groups of bovine embryos, increasing blastocyst rates and decreasing apoptosis rates of these blastocysts.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.