Evaluation of oxytetracycline leaching from pregelatinized starch-coated medicated fish feed.

The rapid growth and intensification of aquaculture industries have led to an increased use of antibiotics. Consequently, growing concerns have mounted over the environmental contamination of these drugs from medicated feeds and the risk that this poses for antimicrobial resistance. To circumvent environmental leaching, farmers topcoat medicated feeds with oil; however, this only partially addresses the issue. This study investigated the potential of food-grade pregelatinized corn starch (PGS) as a second top-coating agent to reduce oxytetracycline (OTC) leaching from the hand-mixed medicated feed. We immersed top-coated medicated feeds for different periods of time and measured the concentration of OTC in the water to determine leaching. We found a significantly lower level of OTC in water samples collected from the PGS-coated medicated feed compared to the non-PGS-coated medicated feed, with concentrations of OTC approximately 4 and 2.6 times the latter after 5 min and 2 h of water immersion, respectively. We also fed PGS-coated antibiotic feed to jade perch to determine if fish accepted the top-coating and whether they absorbed the OTC. Results from a feeding trial suggested no difference in palatability between PGS and non-PGS-coated medicated feed. We also found that muscle tissue from fish fed with the aforementioned diets had similar levels of OTC concentrations, suggesting that PGS coating does not alter the gastrointestinal absorption of this medication. From our experiment, we conclude that PGS is potentially a new top-coating agent to reduce leaching in hand-mixed OTC medicated feed.

Industrial enzymes-producing marine bacteria from marine resources.

Industrial enzymes are important for various biotechnological applications. Currently, the diversity of industrial enzymes-producing marine bacteria from Malaysia remains mostly unknown. This study investigated the diversity of industrial enzyme-producing marine bacteria from culture collections at the Institute of Marine Biotechnology, Universiti Malaysia Terengganu. Out of 200 bacterial isolates revived, 163 bacteria isolate were successfully growth. Marine bacteria produced enzymes with total scoring higher than four were selected for molecular identification using 16S rDNA. About 161 bacteria isolate secreted amylase (68.7 %), lipase (88.3 %) and protease (68.7 %). The phylogenetic analysis led to the identification of three major phyla, namely Proteobacteria, Firmicutes and Bacteroidetes. These phyla were differentiated into nine genera consisted of Bacillus, Chryseomicrobium, Photobacterium, Pseudoalteromonas, Ruegeria, Shewanella, Solibacillus, Tenacibaculum and Vibrio. Genetic variation was more likely to occur within similar marine bacteria species. The microbial community was found to affect the production of industrial enzymes and the diversity of marine bacteria.

Anti-inflammatory effects of Lactobacillus brevis K65 on RAW 264.7 cells and in mice with dextran sulphate sodium-induced ulcerative colitis.

Lactic acid bacteria (LAB) with anti-inflammatory effects may be beneficial to the prevention or treatment for inflammation-related diseases, such as inflammatory bowel diseases. In an in vitro assay, heat-killed Lactobacillus brevis K65 (K65) reduced lipopolysaccharide-induced production of nitric oxide, tumour necrosis factor (TNF)-α and prostaglandin E2 in RAW 264.7 cells. In RAW 264.7 cells stably expressing an ind=ucible nitric oxide synthase (iNOS) reporter, viable K65 showed greater inhibition of iNOS production than its heat-killed form. In order to further examine the in vivo anti-inflammatory effect of K65, viable K65 was orally administered to BALB/c mice before and during the period of dextran sulphate sodium (DSS)-induced ulcerative colitis (UC). K65 improved UC symptoms, including reduced the levels of the pro-inflammatory cytokines, TNF-α, interleukin (IL)-6 and IL-1ß, and lowered the activity of myeloperoxidase. Furthermore, K65 inhibited TNF-α, cyclo-oxygenase 2, forkhead box P3, and Toll-like receptor 4 mRNA expression in the colonic tissue of DSS-induced UC mice. Taken together, K65, a LAB with in vitro anti-inflammatory activity showed preventive effects on mice with DSS-induced UC by lowering the expression of inflammatory molecules.

Epidemic reemergence in adaptive complex networks.

The dynamic nature of a system gives rise to dynamical features of epidemic spreading, such as oscillation and bistability. In this paper, by studying the epidemic spreading in growing networks, in which susceptible nodes may adaptively break the connections with infected ones yet avoid being isolated, we reveal a phenomenon, epidemic reemergence, where the number of infected nodes is incubated at a low level for a long time and then erupts for a short time. The process may repeat several times before the infection finally vanishes. Simulation results show that all three factors, namely the network growth, the connection breaking, and the isolation avoidance, are necessary for epidemic reemergence to happen. We present a simple theoretical analysis to explain the process of reemergence in detail. Our study may offer some useful insights, helping explain the phenomenon of repeated epidemic explosions.

Prostacyclin protects renal tubular cells from gentamicin-induced apoptosis via a PPARalpha-dependent pathway.

To study the protective effect of prostacyclin (PGI2) we increased PGI2 production by infected NRK-52E cells with an adenovirus carrying cyclooxygenase-1 and prostacyclin synthase. PGI2 overexpression protected these cells from gentamicin-induced apoptosis by reducing cleaved caspase-3 and caspase-9, cytochrome c, and decreasing generation of reactive oxygen species. Expression of the nuclear receptor of PGI2, peroxisome proliferator-activated receptor-alpha (PPARalpha), was reduced during gentamicin treatment of the cells, while its overexpression significantly inhibited gentamicin-induced apoptosis and the amount of cleaved caspase-3. Transformation with PPARalpha short interfering RNA abolished the protective effect of PGI2 overproduction in gentamicin-treated cells. The PPARalpha activator docosahexaenoic acid given to gentamicin-treated mice significantly reduced the number of apoptotic cells in renal cortex, but this protective effect was not seen in PPARalpha knockout mice. Our study suggests that increased endogenous PGI2 production protects renal tubular cells from gentamicin-induced apoptosis through a PPARalpha-signaling pathway.

Dynamics of gain-guided solitons in an all-normal-dispersion fiber laser.

We report experimental and numerical results on the dynamics of gain-guided solitons in a passively mode-locked erbium-doped fiber laser made of purely normal dispersion fibers. We show that formation of the soliton in the laser is a result of mutual interaction and balance among the cavity transmission, fiber Kerr nonlinearity, gain saturation, and filtering over one cavity round trip.

Bound states of dispersion-managed solitons in a fiber laser at near zero dispersion.

We report on the observation of various bound states of dispersion-managed (DM) solitons in a passively mode-locked erbium-doped fiber ring laser at near zero net cavity group velocity dispersion (GVD). The generated DM solitons are characterized by their Gaussian-like spectral profile with no sidebands, which is distinct from those of the conventional solitons generated in fiber lasers with large net negative cavity GVD, of the parabolic pulses generated in fiber lasers with positive cavity GVD and negligible gain saturation and bandwidth limiting, and of the gain-guided solitons generated in fiber lasers with large positive cavity GVD. Furthermore, bound states of DM solitons with fixed soliton separations are also observed. We show that these bound solitons can function as a unit to form bound states themselves. Numerical simulations verified our experimental observations.

Generation of multiple gain-guided solitons in a fiber laser.

We report the experimental observation of multiple gain-guided solitons in an erbium-doped fiber laser made of all normal-dispersion fibers. Numerical simulations show that, in the case of a narrow gain bandwidth, under the action of the cavity pulse peak clamping effect multiple gain-guided solitons can indeed be formed in a laser.

Gain-guided solitons in dispersion-managed fiber lasers with large net cavity dispersion.

Gain-guided solitons are experimentally observed in dispersion-managed fiber lasers with large net positive group-velocity dispersion. It is shown that formation of the soliton is a robust feature of the lasers. Numerical simulations also confirmed the experimental results.

Inhibition of angiotensin II induced endothelin-1 gene expression by 17-beta-oestradiol in rat cardiac fibroblasts.

OBJECTIVE: To examine whether 17-beta-oestradiol (E(2)) may alter angiotensin II (Ang II) induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts. DESIGN: Cultured rat cardiac fibroblasts were preincubated with E(2) then stimulated with Ang II. [(3)H]Thymidine incorporation and endothelin-1 (ET-1) gene expression were examined. The effect of E(2) on Ang II induced NADPH oxidase activity, reactive oxygen species (ROS) formation, and extracellular signal regulated kinase (ERK) phosphorylation were tested to elucidate the intracellular mechanism of E(2) in proliferation and ET-1 gene expression. RESULTS: Ang II increased DNA synthesis, which was inhibited with E(2) (1-100 nmol/l). E(2), but not 17-alpha-oestradiol, inhibited Ang II induced ET-1 gene expression as shown by northern blotting and promoter activity assay. This effect was prevented by co-incubation with the oestrogen receptor antagonist ICI 182,780 (1 micromol/l). E(2) also inhibited Ang II increased NADPH oxidase activity, ROS formation, ERK phosphorylation, and activator protein-1 mediated reporter activity. CONCLUSIONS: The results suggest that E(2) inhibits Ang II induced cell proliferation and ET-1 gene expression, partially by interfering with the ERK pathway through attenuation of ROS generation. Thus, this study provides important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of oestrogen on the cardiovascular system.

Supplemention with tetrahydrobiopterin suppresses the development of hypertension in spontaneously hypertensive rats.

It has been suggested that tetrahydrobiopterin (H(4)B), a cofactor of NO synthase, can reverse endothelial dysfunction caused by cardiovascular diseases, including atherosclerosis, coronary artery disease, and hypertension. Moreover, an impairment of H(4)B biosynthesis in spontaneously hypertensive rats (SHR) was observed. Thus, we hypothesized that the defect of the H(4)B synthesis system may play an important role in the development of hypertension in SHR. In the present study H(4)B (10 mg/kg per day IP) was used to treat SHR and Wistar-Kyoto rats (WKY) from the age of 5 through 16 weeks. Results demonstrated that chronic treatment with H(4)B significantly improved the impaired vascular responses to acetylcholine and suppressed the development of hypertension in SHR but did not affect WKY. The increase of inducible NO synthase expression, nitrotyrosine immunostaining, NO production, and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with H(4)B. In contrast, H(4)B had no effect on WKY. In conclusion, this study demonstrated that H(4)B significantly attenuated the development of hypertension in SHR. The antihypertensive effect of H(4)B might be mediated through its direct antioxidant activity and/or decreasing oxygen free radical production from NO synthase, thereby reducing inducible NO synthase expression and peroxynitrite formation. Thus, the present study proposed that supplementation with H(4)B might be beneficial in preventing pathological conditions such as essential hypertension.

Reactive oxygen species mediate cyclic strain-induced endothelin-1 gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in endothelial cells.

Endothelin-1 (Et-1) is a peptide synthesized by endothelial cells (ECs) both in culture and in vivo. Cyclic strain induces gene expression of Et-1, however, the molecular mechanisms remain unclear. Since cyclic strain induces a sustained increase in intracellular reactive oxygen species (ROS), we hypothesized that the ROS could be a modulator in strain-induced Et-1 gene expression. Human umbilical vein ECs (HUVECs) subjected to cyclic strain had increased Et-1 secretion. Pretreatment of HUVECs with antioxidants, catalase (300 U/ml) or 1,3-dimethyl-2-thiourea (DMTU, 0.1 mm), abolished the strain-induced Et-1 release. ECs strained for 6 h had elevated Et-1 mRNA levels. In contrast, ECs treated with catalase or DMTU did not have increase Et-1 mRNA levels stimulated by cyclic strain. Bovine aortic ECs (BAECs) transfected with fusion plasmid containing Et-1 5'-flanking sequence (4.4 kb) and chloramphenicol acetyltransferase reporter gene produced a maximal Et-1 promoter activity after undergoing strain for 6 h, whereas pretreatment with catalase decreased this activity. BAECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or catalytically inactive mutant of extracellular signal-regulated kinase (mERK2) had inhibited strain-induced Et-1 promoter activity, indicating the Ras/Raf/ERK pathway was involved; moreover, ERK phosphorylation was induced in ECs which were strained. This strain-activated ERK phosphorylation was attenuated in the presence of catalase. Functional analysis of the Et-1 promoter with site-directed mutagenesis indicates that the activator protein-1 (AP-1) binding site had to be within 143 base-pairs upstream of transcription initiation site for strain-induced promoter activity. Pretreatment of ECs with catalase also decreased the strain-induced promoter activity in the minimal construct (-143 bp). Our data demonstrate that strain-induced Et-1 gene expression is modulated by ROS via Ras/Raf/ERK signaling pathway, and indicate the responsiveness of the AP-1 binding site for strain-induced Et-1 expression.

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Reactive oxygen species modulate angiotensin II-induced beta-myosin heavy chain gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in neonatal rat cardiomyocytes.

Angiotensin II (Ang II) causes cardiomyocytes hypertrophy. Cardiac beta-myosin heavy chain (beta-MyHC) gene expression can be altered by Ang II. The molecular mechanisms are not completely known. Reactive oxygen species (ROS) are involved in signal transduction pathways of Ang II. However, the role of ROS on Ang II-induced beta-MyHC gene expression remains unclear. Here we found that Ang II increased beta-MyHC promoter activity and it was blocked by Ang II type 1 receptor antagonist losartan. Ang II dose-dependently increased the intracellular ROS. Cardiomyocytes cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal regulated kinase (mERK2) inhibited Ang II-induced beta-MyHC promoter activity, indicating Ras/Raf/ERK pathway was involved. Antioxidants such as catalase or N-acetyl-cysteine decreased Ang II-activated ERK phosphorylation and inhibited Ang II-induced beta-MyHC promoter activity. These data indicate that Ang II increases beta-MyHC gene expression in part via the generation of ROS.

Prokinetic agents increase plasma albumin in hypoalbuminemic chronic dialysis patients with delayed gastric emptying.

Hypoalbuminemia is a surrogate of malnutrition in patients with end-stage renal disease undergoing chronic dialysis and commonly improves with prescription of adequate nutrition and dialysis. Nevertheless, some patients remain hypoalbuminemic for poorly understood reasons. We tested the hypotheses that chronic dialysis patients who remain hypoalbuminemic despite prescription of adequate nutrition and dialysis (1) have delayed gastric emptying, and (2) that prokinetic agents will increase plasma albumin (P(alb)) levels in patients with delayed gastric emptying. We retrospectively identified 99 of 343 hemodialysis and peritoneal dialysis patients with hypoalbuminuria (P(alb) < 3.5 mg/dL) and studied those who did not (hypoalbuminemic, n =15) and did (normoalbuminemic, n = 15) increase their P(alb) levels over the subsequent 6 months and met inclusion and exclusion criteria. Gastrointestinal symptom scores determined by an administered questionnaire were not different in hypoalbuminemic and normoalbuminemic patients. Conversely, the half-time (T(1/2)) for radionuclide gastric emptying was longer in hypoalbuminemic than normoalbuminemic patients (74.5 +/- 7.4 versus 46.7 +/- 4.6 minutes; P < 0.004). Hypoalbuminemic patients were prescribed prokinetics and followed prospectively for 6 months, during which time gastric T(1/2) decreased to 53.9 +/- 3.3 minutes (P < 0.01 versus initial) and P(alb) increased from 3.1 +/- 0.2 to 3.5 +/- 0.2 mg/dL (P < 0.004). The net increase in P(alb) level correlated with the net decrease in gastric T(1/2) (r(2) = 0.4; P < 0.04) by linear regression. The data show that some persistently hypoalbuminemic chronic dialysis patients have poor gastric emptying and increase their P(alb) levels in response to prokinetic agents.

Establishment of transcriptional silencing in the absence of DNA replication.

Transcriptional repression of the silent mating-type loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commonly assumed to involve DNA replication. Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of replication in establishment of silencing. Sir1 was tethered to the ring following excision from the chromosome to activate a dormant silencer. We show here that silencing can be established in DNA that does not replicate. The silenced ring adopted structural features characteristic of bona fide silent chromatin, including an altered level of DNA supercoiling and reduced histone acetylation. In addition, the process required silencing factors Sir2, Sir3, and Sir4 and progression between early S and M phases of the cell cycle. The results indicate that passage of a replication fork is not the cell-cycle event required for establishment of silencing in yeast.

Yeast heterochromatin is a dynamic structure that requires silencers continuously.

Transcriptional silencing of the HM loci in yeast requires cis-acting elements, termed silencers, that function during S-phase passage to establish the silent state. To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembled silent chromatin fragments from silencers. DNA rings excised from HMR were initially silent but ultimately reactivated, even in G(1)- or G(2)/M-arrested cells. In contrast, DNA rings bearing HML-derived sequence were stably repressed due to the presence of a protosilencing element. These data show that silencers (or protosilencers) are required continuously for maintenance of silent chromatin. Reactivation of unstably repressed rings was blocked by overexpression of silencing proteins Sir3p and Sir4p, and chromatin immunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin. Importantly, the protein was incorporated even when expressed outside of S phase, during G(1) arrest. That silencing factors can associate with and stabilize preassembled silent chromatin in non-S-phase cells demonstrates that heterochromatin in yeast is dynamic.

Controlling gene expression in yeast by inducible site-specific recombination.

An intron module was developed for Saccharomyces cerevisiae that imparts conditional gene regulation. The kanMX marker, flanked by loxP sites for the Cre recombinase, was embedded within the ACT1 intron and the resulting module was targeted to specific genes by PCR-mediated gene disruption. Initially, recipient genes were inactivated because the loxP-kanMX-loxP cassette prevented formation of mature transcripts. However, expression was restored by Cre-mediated site-specific recombination, which excised the loxP-kanMX-loxP cassette to generate a functional intron that contained a single loxP site. Cre recombinase activity was controlled at the transcriptional level by a GAL1::CRE expression vector or at the enzymatic level by fusing the protein to the hormone-dependent regulatory domain of the estrogen receptor. Negative selection against leaky pre-excision events was achieved by growing cells in modified minimal media that contained geneticin (G418). Advantages of this gene regulation technique, which we term the conditional knock-out approach, are that (i) modified genes are completely inactivated prior to induction, (ii) modified genes are induced rapidly to expression levels that compare to their unmodified counterparts, and (iii) it is easy to use and generally applicable.

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes.

OBJECTIVES: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. METHODS: Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. RESULTS: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. CONCLUSIONS: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo.

A burgeoning interest in the role of chromatin structure in a wide variety of chromosome functions has established a need for methods to obtain chromatin in its native form. Here we describe a simple and efficient method for biochemical isolation of selected chromatin fragments from yeast chromosomes. The approach involves three steps. First, site-specific recombination in vivo is used to excise a chromosomal domain of interest in the form of a small extrachromosomal ring. Second, whole cell lysate is prepared from cultures in which recombination has been induced. Third, differential centrifugation is used to separate excised chromatin rings from chromosomes and other cellular debris. Using this methodology, we show that rings containing the transcriptionally repressed HMR mating-type locus can be formed and isolated in high yield. Furthermore, we show that the isolation procedure results in significant enrichment of recombinant rings. Finally, we show that the nucleosomal organization of the recombined material is not altered during isolation.