Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.     

半胱氨酸血红蛋白配合物着色剂的研究

运用分子轨道杂化理论和配合物晶体场理论论证了半胱氨酸和血红蛋白之间发生配位反应的可能性。以新鲜猪血中的血红蛋白为原料,与半胱氨酸反应制备半胱氨酸血红蛋白配合物发色剂。对产物进行紫外、红外光谱表征,证明生成的新物质是半胱氨酸血红蛋白配合物。通过单因素和正交试验,以溶液在540nm处吸光度为指标,考察pH、温度、反应时间和半胱氨酸添加量的影响,试验确定半胱氨酸与血红蛋白配位反应的最佳条件为pH7.5、温度25℃、反应时间1min、半胱氨酸添加量0.4%。通过灌肠实验考察了新型无硝发色剂在实际运用中的效果。     

Transformation of the recalcitrant pharmaceutical compound carbamazepine by Pleurotus ostreatus: role of cytochrome P450 monooxygenase and manganese peroxidase

Carbamazepine (CBZ) is an environmentally recalcitrant compound highly stable in soil and during wastewater treatment. In this study, we examined the mechanisms by which the white-rot fungus Pleurotus ostreatus metabolizes CBZ in liquid culture using a physiological approach. P. ostreatus PC9 was grown in media known to support different levels of a multiplicity of enzyme systems such as cytochrome P450 (CYP450) and manganese peroxidase (MnP). When both CYP450 and MnP systems were active, 99% of the added CBZ was eliminated from the solution and transformed to 10,11-epoxycarbamazepine. High removal of CBZ was also obtained when either MnP or CYP450 was active. When both CYP450 and MnP were inactivated, only 10 to 30% of the added CBZ was removed. In this latter system, removal of CBZ might be partially attributed to the activity of versatile peroxidase. P. ostreatus was able to eliminate CBZ in liquid culture even when CBZ was added at an environmentally relevant concentration (1 μg L(-1)). On the basis of our study, we suggest that two families of enzymes are involved in the oxidation of CBZ in liquid culture: MnP in a Mn(2+)-dependent or independent manner and CYP450. Our study also highlights the potential of using P. ostreatus for bioremediation systems.     

In silico guided pre-treatment of sugarcane juice with natural inhibitors of polyphenol oxidase (PPO) is a new and effective strategy for development of spray-dried formulation

Sugarcane juice is a popular beverage and is also processed to produce sugar. The polyphenol oxidase (PPO) in sugarcane juice causes enzymatic browning and makes the process of sugar production complex and cumbersome. Storage of sugarcane juice is also hampered by the high sugar content and rapid microbial fermentation. The present research assessed the potential of lemon juice (LJ) and ginger extract (GE) as natural inhibitors of PPO. Enzyme kinetics and the mechanism of inhibition of LJ and GE were studied. Primary investigation was carried out using molecular docking approach to assess the inhibitory potential of LJ and GE and to determine the nature of interaction between the enzyme and inhibitors. Extracts were used as inhibitors and studies revealed that both reduced the PPO activity. Subsequently, pure bioactive inhibitors such as ascorbic acid, citric acid, and 6-shogaol present in these natural extracts were used to study the mode of inhibition of PPO. Citric acid decreased PPO activity by lowering pH, while ascorbic acid was found to be a competitive inhibitor of PPO with a K_i of 75.69 µM. The proportion of LJ and GE required in sugarcane juice was optimized on the basis of browning index and sensory acceptance. Further, the sugarcane cane juice after inhibition of PPO under optimized conditions was spray dried and evaluated for reconstitution properties. The product formulated in the present study is a new and effective approach to address quality-compromising issues associated with long-term storage of cane juice.     

Purification and characterization of olive (Olea europaea L.) peroxidase – Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18–20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 °C, and apparent K_m values of 41.0 and 0.53 mM, for phenol and H₂O₂, respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 °C, 411.2 and −13.6 kJ mol⁻¹ K⁻¹. The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol⁻¹, corresponding to a calculated temperature coefficient (Q₁₀) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase.     

Boesenbergin A,a chalcone from Boesenbergia rotunda induces apoptosis via mitochondrial dysregulation and cytochrome c release in A549 cells in vitro: Involvement of HSP70 and Bcl2/Bax signalling pathways

The anti-cancer effect of Boesenbergin A (BA) isolated from Boesenbergia rotunda, via the induction of apoptosis resulting from mitochondrial dysfunction was assessed in human non-small cell lung cancer (A549) cells. The apoptotic mechanisms of BA induction on cancer cells were studied in the present study for the first time. Nuclear stain, measuring the accumulation of sub-G₁ cell population and DNA ladder were done to determine the apoptosis. Further investigations into the depletion of mitochondrial membrane potential and release of cytochrome c determined that BA treatment induced apoptosis via the regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The involvement of both intrinsic and extrinsic caspases (caspase 3/7, 9 and 8) were significantly increased. Moreover the role of free radicals was significantly found to be elevated with concomitant decrease in HSP70. In conclusion the results from the current study indicated BA could be a promising agent for the treatment of lung cancer.     

Properties of peroxidase and polyphenol oxidase in natural complexes from walnuts (Juglans regia) and in active DL-DOPA copolymers

Black polymers were extracted from walnut husk homogenates (Juglans regia L) in a phosphate buffer (pH 8.0) after air-bubbling for 24 h. After several purification stages, ultrafiltration and gel chromatography (Sephadex and Ultrogel), three extracts (two soluble and one insoluble) were isolated. At the same time, a synthetic DL DOPA/POD/PPO copolymer was prepared. Two walnut extracts exhibited POD and PPO activity. Both DL DOPA/POD/PPO copolymers (soluble and insoluble) only presented a POD activity. The insoluble and more complex extracts (either natural or synthetic) presented the lowest oxidase activities. The soluble walnut extract exhibited low though not negligible PPO and POD activities. It was observed using electrophoresis and gel chromatography that both enzymes were present in phenolic complexes. The high level of adsorption observed on polyacrylamide gel, as well as the elementary composition of these complexes enabled a comparison with phytomelanins. PPO and POD in the soluble copolymers (from both natural and synthetic sources) were governed by Michaelis-Menten kinetics. Apparent constants were calculated using the Lineweaver-Burk method. The use of limited proteolysis and an inhibitor showed good resistance of POD activity in walnut and synthetic copolymers and exceptional resistance of PPO activity in walnut extracts (particularly insoluble extract).     

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84 ± 1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.     

Characterization of the cytochrome c gene from the starch-fermenting yeast Schwanniomyces occidentalis and its expression in Baker's yeast

A cytochrome c protein gene, CYC10, of the dextran- and starch-fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein-coding region shares close homologies to the cytochrome c genes. A S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein-encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiae. The CYC10 gene was oxygen-induced but not subject to catabolite repression. The expression of the CYC10 gene was studied in the heterologous yeast S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indicating that these two genes share similar or closely related cis- and trans-acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. occidentalis cells. Search in the 5' untranslated region of the CYC10 gene revealed some homologies at -425 to -405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae. Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.     

A new strategy for the analysis of tetracycline residues in foodstuffs by a surface plasmon resonance biosensor

A new strategy was developed for the establishment of an indirect tetracycline assay using surface plasmon resonance (SPR). The principle for the assay is based on the most important resistance mechanism against tetracycline in gram-negative bacteria. Tetracyclines release the 46.6 kDa Tet repressor protein (TetR) from the tet operator (tetO), a biotinylated short double strand DNA sequence bound to a streptavidin biosensor chip. Tetracyclines present in a sample solution bind to the repressor protein, inducing a conformational change accompanied with a reduction of the affinity constant of TetR to tetO. We were able to quantify tetracycline residues in spiked raw milk samples in concentrations corresponding to the maximum residue limit (MRL) set by the European Union. Honey samples could also be analysed but with lower sensitivity. The assay detects seven of the most commonly used tetracyclines in veterinary medicine. Nine antibiotics of five other antibiotic classes were tested and no interference with the SPR assay was found. Thus, the described principle forms the basis for screening assays to routinely test for tetracycline residues in foodstuffs.     

我国茶业发展的新成就、新思路和新举措

本文叙述了我国茶业发展的新成就;茶业持续发展的新思路;今后加快茶业发展的新举措。     

Co-amplification and sequencing of a cytochrome b fragment affecting the identification of cattle in PCR-RFLP food authentication studies

Food authentication studies based on PCR-RFLP analysis are frequently targeted to well-conserved mitochondrial sequences, such as certain regions of the cytochrome b (cytb) gene. The use of mitochondrial L14841/H15149 universal cytb-targeted primers in PCR-RFLP assays revealed the existence of a complex restriction pattern in three genetically-unrelated Iberian (Northern Spain) cows, this being due to the simultaneous co-amplification of two 359 bp cytb fragments. Microsatellite analysis of 11 bovine-specific loci confirmed no familiar linkage among the animals investigated. Both co-amplification products were successfully separated by specific cleavage with endonucleases RsaI and MvaI, which allowed the recovery of each amplification product, respectively. The new co-amplified cytb fragment described in this study was successfully sequenced, and exhibited a significantly high homology (95.1–99.3% range) with respect to mitochondrial sequences previously described for a Bos indicus specimen and for another two Asian Bos taurus, this underlining that its presence in cattle may be more extended than initially thought. In contrast, the homology with the cytb sequence widely accepted for B. taurus was only 89.6%. The results presented in this work imply that food authentication studies by PCR-RFLP analysis may be complicated in the case of cattle by the co-amplification of two different cytb fragments.     

LC-MS/MS analytical procedure to quantify tris(nonylphenyl)phosphite,as a source of the endocrine disruptors 4-nonylphenols,in food packaging materials

Tris(nonylphenyl)phosphite, an antioxidant used in polyethylene resins for food applications, is problematic since it is a source of the endocrine-disrupting chemicals 4-nonylphenols (4NP) upon migration into packaged foods. As a response to concerns surrounding the presence of 4NP-based compounds in packaging materials, some resin producers and additive suppliers have decided to eliminate TNPP from formulations. This paper describes an analytical procedure to verify the “TNPP-free” statement in multilayer laminates used for bag-in-box packaging. The method involves extraction of TNPP from laminates with organic solvents followed by detection/quantification by LC-MS/MS using the atmospheric pressure chemical ionisation (APCI) mode. A further acidic treatment of the latter extract allows the release of 4NP from potentially extracted TNPP. 4NP is then analysed by LC-MS/MS using electrospray ionisation (ESI) mode. This two-step analytical procedure ensures not only TNPP quantification in laminates, but also allows the flagging of other possible sources of 4NP in such packaging materials, typically as non-intentionally added substances (NIAS). The limits of quantification were 0.50 and 0.48 µg dm^–2 for TNPP and 4NP in laminates, respectively, with recoveries ranging between 87% and 114%. Usage of such analytical methodologies in quality control operations has pointed to a lack of traceability at the packaging supplier level and cross-contamination of extrusion equipment at the converter level, when TNPP-containing laminates are processed on the same machine beforehand.     

Izumoring: a novel and complete strategy for bioproduction of rare sugars

Starch, whey or hemicellulosic waste can be used as a raw material for the industrial production of rare sugars. D-glucose from starch, whey and hemicellulose, D-galactose from whey, and D-xylose from hemicellulose are the main starting monosaccharides for production of rare sugars. We can produce all monosaccharides; tetroses, pentoses and hexoses, from these raw materials. This is achieved by using D-tagatose 3-epimerase, aldose isomerase, aldose reductase, and oxidoreductase enzymes or whole cells as biocatalysts. Bioproduction strategies for all rare sugars are illustrated using ring form structures given the name Izumoring.     

新时期环境监察执法现状与提升策略

近年来,随着经济的高速运转,人们物质生活水平不断提升,经济发展导致各种环境污染问题日趋严重.为了更好地服务绿色经济,环境监察部门扮演着不可或缺的重要角色.环境监察部门要在适应新时代要求的前提下,持续更新执法理念、优化执法效果、充分担责履职,在经济发展的同时助力环境保护工作逐步落实.本文从我国环境监察执法工作的现状为切入...     

A new strategy for improved glutathione production from Saccharomyces cerevisiae: use of cysteine‐ and glycine‐rich chicken feather protein hydrolysate as a new cheap substrate

BACKGROUND: Glutathione (GSH) is composed of the amino acids glutamic acid, cysteine and glycine. This study investigated the usability of chicken feather protein hydrolysate (chicken feather peptone, CFP) as a substrate for GSH production from Saccharomyces cerevisiae. RESULTS: CFP was found to be rich in ash (36.7 g per 100 g), protein (61.1 g per 100 g) and minerals (S, P, K, Ca, Fe, Na and Mg). It also had high contents of cysteine and glycine. CFP augmented biomass and GSH production by 53 and 115% respectively compared with the control medium. The highest biomass (17.4 g l^?1) and GSH (271 mg L^?1) concentrations were attained in CFP medium. The second highest biomass (16.8 g l^?1) and GSH (255 mg L^?1) concentrations were obtained in fish peptone medium. It was assumed that the high mineral, cysteine and glycine contents of CFP were related to cell growth and GSH synthesis in S. cerevisiae. CONCLUSION: This is the first report on the effect of cysteine‐ and glycine‐rich protein hydrolysates on GSH production from S. cerevisiae. In this regard, CFP was tested for the first time as a GSH production substrate. As an additional contribution, a new hydrolysis process was developed for the preparation of protein hydrolysates. © 2012 Society of Chemical Industry     

Differentiation of chicken gonad as an endocrine organ: expression of LH receptor,FSH receptor,cytochrome P450c17 and aromatase genes

The gonad is an endocrine organ secreting sex hormones and also a target of pituitary gonadotrophins. The expression of mRNAs encoding LH receptor (LHR), FSH receptor (FSHR), P450c17 and P450aromatase in the developing gonads of embryos between day 4 and day 6 of incubation was determined using a RT-PCR to elucidate the chicken gonad as a target organ of gonadotrophins. Although expression of mRNAs encoding LHR, FSHR and P450c17 was detected at day 4 of incubation in both sexes, mRNA encoding P450aromatase appeared at day 6 in female embryos only, indicating that mRNAs encoding gonadotrophin receptors can be identified before sexual differentiation. Quantitative PCR analysis revealed that expression of mRNA encoding LHR and FSHR remained low in male gonads from day 4 to day 6 of incubation, whereas they increased on day 6 in female gonads. The sexual dimorphism in the expression of mRNAs encoding LHR and FSHR was confirmed in the sexually differentiated gonads of embryos at day 12 of incubation (LHR in ovary ratio LHR in testis = 7 ratio 1; FSHR in ovary ratio FSHR in testis = 9 ratio 1).     

Optimisation of a coupled enzymatic assay of glutathione peroxidase activity in bovine milk and whey

Glutathione peroxidase (GSHPx) may have important functions in milk but studies of its activity during dairy processing are hampered by the lack of suitable assays. Thus, a coupled enzymatic assay of GSHPx activity was developed for bovine milk and whey. The reaction mixture contained reduced glutathione, tert-butylhydroperoxide, glutathione reductase and NADPH in phosphate buffer. The effect of reaction conditions on enzymatic and non-enzymatic NADPH consumption was studied, and blanks without added GSHPx sample or complete incubations containing 4 mmol L⁻¹ mercaptosuccinate as an inhibitor of GSHPx were used. With increasing pH and glutathione concentration the reaction rates increased both in the complete incubation and the blank but optimal GSHPx activity was attained at a glutathione concentration of 0.63 mmol L⁻¹. A pH of 7.6 was chosen to attain near-optimal activity without reaching high blanks. Application of the new procedure showed that bulk whey samples had rather constant GSHPx activity with an average (SD) of 36.1 (2.9) U mL⁻¹, but whey samples prepared from individual cows showed a two-fold variation (range 24.5–50.6 U mL⁻¹). The procedure described was more reproducible and precise than previous methods and will be employed in further studies of dairy products.