Evaluation of culture medium for nisin production in a repeated-batch biofilm reactor

In this study, a biofilm reactor with plastic composite support (PCS), made by high-temperature extrusion of agricultural products and polypropylene, was evaluated for nisin production using L. lactis strain NIZO 22186. The high-biomass density of the biofilm reactor was found to contribute to a significantly shorter lag time of nisin production relative to a suspended-cell reactor. In comparison to glucose (579 IU/mL), sucrose significantly increased the nisin production rate by 1.4-fold (1100 IU/mL). However, results revealed that high levels of sucrose (8% w/v) had a suppressing effect on nisin production and a stimulating effect on lactic acid production. A high concentration of MgSO4.7H2O at 0.04% (w/v) was found to reduce the nisin production, while concentrations of KH2PO4 of up to 3% (w/v) did not have any significant effect on growth or nisin production. The best of the tested complex media for nisin production using the PCS biofilm reactor consisted of 4% (w/v) sucrose, 0.02% (w/v) MgSO4.7H2O, and 0.1% (w/v) KH2PO4. Nisin production rate in the biofilm reactor was significantly increased by 3.8-fold (2208 IU/mL) when using the best complex medium tested.     

Changes of nitric oxide synthase-containing nerve fibers and parameters for oxidative stress after unilateral cavernous nerve resection or manuplation in rat penis

After pelvic surgeries such as radical prostatectomy, two major complications--urinary incontinence and erectile dysfunction (ED) may occur. Etiologies for ED are multiple pathologic mediators/systems. Oxidative stress, which is known to be induced after surgical trauma, could be a cause of ED. The purposes of in this study are to investigate the effect of unilateral manipulation/ dissection and resection of the cavernous nerve (neurotomy) to NOS (nitric oxide synthase)-containing nerve fibers and pressure after electro stimulation in rat corpus cavernosum, and to determine whether these procedures would produce oxidative stress within rat cavernous tissue 3 weeks and 6 months after the operation. Male rats were divided into 5 groups. Rats in groups 1 and 2 underwent unilateral cavernous nerve manipulation and sacrificed 3 weeks and 6 months after the operation, respectively. Rats in groups 3 and 4 underwent unilateral neurotomy of a 5-mm. segment of the cavernous nerve, and they were sacrificed 3 weeks and 6 months after nerve ablation, respectively. Group 5 rats were control animals for biochemical analysis. Intracavernous pressure following electro stimulation reduced is significantly 3 weeks after unilateral resection, as compared to that of the manipulated nerve (P < 0.05), and it recovered 6 months after neurotomy. The recovery was also confirmed by NADPH (nicotinamide adenine dinucleotide phosphate) diaphorase staining in neurotomy groups. Lipid peroxidation, which is an indicater of oxidative stress, was determined by measuring thiobarbituric acid reacting substance (TBARS) levels and superoxide dismutase (SOD) activity. These markers indicated that unilateral cavernous nerve manipulation or resection produced oxidative stress within rat corpus cavernosum. Oxidative stress was more prominent 3 weeks after unilateral neurotomy (P < 0.05). Also, compared to the control animal group, oxidative stress was observed three weeks after manipulation of unilateral cavernous nerve (P < 0.05). Resection of the cavernous nerve caused more prominent oxidative stress than in the manipulation group. This study suggested, that unilateral cavernous neurotomy caused a decrease of intra cavernous pressure and NOS fibers in rat corpus cavernosum, and they recovered 6 months after neurotomy. Our data also provided evidence that neurotomy and manipulation of the cavernous nerve caused oxidative stress in rat corpus cavernosum and that oxidative stress was more prominent in the nerve resection group.     

Inhibition of inducible nitric oxide synthase in murine visceral larva migrans: effects on lung and liver damage

The roles of nitric oxide production and oxidative process were studied in mice infected with Toxocara canis and treated with aminoguanidine which is a specific inhibitor of inducible nitric oxide synthase (iNOS). Relations of nitric oxide synthase inhibition and tissue pathology were assessed by biochemical, histological and immunohistochemical methods. In experiments, Balb/c albino mice were inoculated with T. canis eggs either with or without aminoguanidine treatment or alone, at 24th, 48th hours and on 7th days. LPx and SOD values in liver tissue and plasma were measured. Liver and lung tissues were evaluated for the pathological lesions. The expression of eNOS and iNOS in both tissues were studied with immunohistochemistry in the same intervals. We observed significant differences between T. canis infected and aminoguanidine treated animals. Larval toxocarosis led to oxidative stress elevation in plasma. Microscopic examination of the liver histological sections revealed pathological lesions in the hepatic parenchyma in infected mice. In the mice received T. canis eggs plus aminoguanidine, the sinusoidal areas were enlarged. Histological lesions were more severe at 48 hours after infection. Numbers of eNOS and iNOS expressing epithelial cells were increased in the T. canis infected mice. The activities of eNOS and iNOS were also observed in the body of the larvae which have migrated to lung and liver. As a result, we have demonstrated that in vivo production of eNO and iNO during T. canis infection cause direct host damages and it is strongly related to the oxidative stress. We propose that larval NO can also be effective in larval migration, but it needs further investigation on distribution of NO in larvae.     

L-Cysteine influx and efflux: a possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied L-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of L-cysteine and then measured the intracellular free -SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free -SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free -SH depletion on L-cysteine transport is also investigated. We also determined the rate of L-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our L-cysteine influx studies demonstrated that erythrocytes can respond to increases in L-cysteine concentration in the extracellular media and influx L-cysteine in a concentration-dependent manner. Free -SH concentrations in erythrocytes treated with 1 mM L-cysteine reached to 1.64 +/- 0.06 mM in 1 h whereas this concentration reached to 4.30 +/- 0.01 mM in 10 mM L-cysteine treated erythrocytes. The L-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher L-cysteine concentrations displayed a higher efflux process. Outside concentration of free -SH in 1 mM L-cysteine pretreated erythrocytes reached to 0.200 +/- 0.005 mM in 1 h whereas this concentration reached to 1.014 +/- 0.002 with 10 mM L-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of L-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the L-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the L-cysteine efflux.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

Efflux of Glutathione and Glutathione Complexes from Human Erythrocytes in Response to Inorganic Arsenic Exposure

The objective of the present study was to investigate if arsenic exposure results in glutathione efflux from human erythrocytes. Arsenite significantly depleted intracellular nonprotein thiol level in a time- and concentration-dependent manner. The intracellular nonprotein thiol level was decreased to 0.767?±?0.0017???mol/ml erythrocyte following exposure to 10?mM of arsenite for 4?h. Extracellular nonprotein thiol level was increased concomitantly with the intracellular decrease and reached to 0.481?±?0.0005???mol/ml erythrocyte in 4?h. In parallel with the change in extracellular nonprotein thiol levels, significant increases in extracellular glutathione levels were detected. Extracellular glutathione levels reached to 0.122?±?0.0013, 0.226?±?0.003, and 0.274?±?0.004???mol/ml erythrocyte with 1, 5, and 10?mM of arsenite, respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 and verapamil, multidrug resistance-associated protein 1 and Pgp inhibitors, decreased the rate of glutathione efflux from erythrocytes suggesting a role for these membrane transporters in the process. The results of the present study indicate that human erythrocytes efflux glutathione in reduced free form and in conjugated form or forms that can be recovered with dimercaptosuccinic acid when exposed to arsenite.     

Biotransformation of (-)-(R)-α-Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α-phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6-hydroxypiperitone, α-phellandrene epoxide, cis-p-menth-2-en-1-ol, and carvotanacetone, which originated from (-)-(R)-α-phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5-p-menthene-1,2-diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities (MICs=0.125 to >4?mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (-)-(R)-α-phellandrene and standard antimicrobial agents.     

Monitoring a bioprocess for ethanol production using FT-MIR and FT-Raman spectroscopy

The application of Fourier transform mid-infrared (FT-MIR) spectroscopy and Fourier transform Raman (FT-Raman) spectroscopy for process and quality control of fermentative production of ethanol was investigated. FT-MIR and FT-Raman spectroscopy along with multivariate techniques were used to determine simultaneously glucose, ethanol, and optical cell density of Saccharomyces cerevisiae during ethanol fermentation. Spectroscopic measurement of glucose and ethanol were compared and validated with the high-performance liquid chromatography (HPLC) method. Spectral wave number regions were selected for partial least-squares (PLS) regression and principal component regression (PCR) and calibration models for glucose, ethanol, and optical cell density were developed for culture samples. Correlation coefficient (R ²) value for the prediction for glucose and ethanol was more than 0.9 using various calibration methods. The standard error of prediction for the PLS first-derivative calibration models for glucose, ethanol, and optical cell density were 1.938 g/l, 1.150 g/l, and 0.507, respectively. Prediction errors were high with FT-Raman because the Raman scattering of the cultures was weak. Results indicated that FT-MIR spectroscopy could be used for rapid detection of glucose, ethanol, and optical cell density in S. cerevisiae culture during ethanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 185–190. Received 16 November 2000/ Accepted in revised form 12 January 2001