Protective effects of protocatechuic acid on growth performance, intestinal barrier and antioxidant capacity in broilers challenged with lipopolysaccharide.

'Prohibition of the antibiotic uses' aggravates the problem of intestinal diseases in poultry, and nutritional regulation has become a research hotspot, such as supplementation with active ingredients derived from plants. This research was conducted to investigate the effects of protocatechuic acid (PCA) on growth, intestinal barrier, and antioxidant capacity of broilers injected with lipopolysaccharide (LPS). Four hundred and eighty 1-day-old yellow feather broilers were randomly allocated to four groups, each with six replicates of 20 broilers. The treatments were basal diet + saline injection (CON) or LPS injection (CON-LPS), and diets with 300 or 600 mg/kg PCA supplementation + LPS injection (P300, P600). Birds were injected intramuscularly on 17th and 19th day of age, then sampled on day 21. The LPS injection significantly decreased BW and average daily gain of broilers, and compared with birds in CON-LPS, PCA supplementation increased (P < 0.05) those variables; moreover, 300 mg/kg PCA also decreased the feed-to-gain ratio. No differences were observed in relative weights of immune organs (P > 0.05). LPS decreased the villus height/crypt depth ratio (V/C) in jejunum of broilers, while PCA (P300 and P600) increased (P < 0.05) the jejunal villus height and V/C compared with birds in CON-LPS. LPS challenge increased jejunal malondialdehyde (MDA) concentration and decreased total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities in plasma (P < 0.05); compared with birds in CON-LPS, jejunal and plasmal GSH-Px activity (P300 and P600) and jejunal T-SOD activity (P300) were decreased (P < 0.05), and hepatic MDA concentration (P600) was increased (P < 0.05). LPS significantly decreased the transcript abundances of OCLN, ZO-1, JAM2, MUC2, SOD1, CAT and GPX in jejunal mucosa of birds, and supplementation with PCA attenuated the decrease in OCLN, JAM2, and MUC2 expression compared with birds in CON-LPS; moreover, 600 mg/kg PCA offset the deduction in SOD1, CAT and GPX expression. In conclusion, dietary supplementation with PCA could improve antioxidant status and attenuate the damage in intestinal barrier and loss in growth performance of LPS-challenged broilers, and 600 mg/kg PCA showed more improved effects on antioxidant capacity.

Effects of dietary incorporation of linseed oil with soybean isoflavone on fatty acid profiles and lipid metabolism-related gene expression in breast muscle of chickens.

The meat quality of chicken is an important factor affecting the consumer's health. It was hypothesized that n-3 polyunsaturated fatty acid (n-3 PUFA) could be effectively deposited in chicken, by incorporating antioxidation of soybean isoflavone (SI), which led to improved quality of chicken meat for good health of human beings. Effects of partial or complete dietary substitution of lard (LA) with linseed oil (LO), with or without SI on growth performance, biochemical indicators, meat quality, fatty acid profiles, lipid-related health indicators and gene expression of breast muscle were examined in chickens. A total of 900 males were fed a corn-soybean meal diet supplemented with 4% LA, 2% LA + 2% LO and 4% LO and the latter two including 30 mg SI/kg (2% LA + 2% LO + SI and 4% LO + SI) from 29 to 66 days of age; each of the five dietary treatments included six replicates of 30 birds. Compared with the 4% LA diet, dietary 4% LO significantly increased the feed efficiency and had no negative effect on objective indices related to meat quality; LO significantly decreased plasma triglycerides and total cholesterol (TCH); abdominal fat percentage was significantly decreased in birds fed the 4% LO and 4% LO + SI diets. Chickens with LO diets resulted in higher contents of α-linolenic acid (C18:3n-3), EPA (C20:5n-3) and total n-3 PUFA, together with a lower content of palmitic acid (C16:0), lignoceric acid (C24:0), saturated fatty acids and n-6:n-3 ratio in breast muscle compared to 4% LA diet (P < 0.05); they also significantly decreased atherogenic index, thrombogenic index and increased the hypocholesterolemic to hypercholesterolemic ratio. Adding SI to the LO diets enhanced the contents of EPA and DHA (C22:6n-3), plasma total superoxide dismutase, reduced glutathione (GSH)/oxidized glutathione and muscle GSH content, while decreased plasma total triglyceride and TCH and malondialdehyde content in plasma and breast muscle compared to its absence (P < 0.05). Expression in breast muscle of fatty acid desaturase 1 (FADS1), FADS2, elongase 2 (ELOVL2) and ELOVL5 genes were significantly higher with the LO diets including SI than with the 4% LA diet. Significant interactions existed between LO level and inclusion of SI on EPA and TCH contents. These findings indicate that diet supplemented with LO combined with SI is an effective alternative when optimizing the nutritional value of chicken meat for human consumers.

Dietary curcumin enhances intestinal antioxidant capacity in ducklings via altering gene expression of antioxidant and key detoxification enzymes.

The study investigated the effects of dietary curcumin supplementation on tissue distribution of curcumin and its metabolites, intestinal antioxidant capacity, and expression of detoxification-related genes in ducks. A total of 720 one-day-old male Cherry Valley Pekin ducklings (initial BW 58.6 ± 0.1 g) were randomly assigned to 4 dietary groups each with 6 replicates of 30 ducks using a single factorial arrangement design. Ducks in the control group were fed a basal diet and the remainder were fed the basal diet supplemented with 200, 400, or 800 mg/kg curcumin. The experiment lasted for 21 D. Curcumin was present at 13.12 to 16.18 mg/g in the cecal digesta, 75.50 to 575.40 µg/g in jejunal mucosa, 35.10 to 73.65 µg/g in liver, and 7.02 to 7.88 µg/mL in plasma. The jejunal and hepatic contents of curcumin increased significantly (P < 0.05) in response to supplementation with 400 and 800 mg/kg of curcumin respectively, compared with 200 mg curcumin/kg group. There was a linear (P < 0.001) effect of dietary curcumin on relative abundance of SOD1, GPX1, CAT, HO-1, and Nrf2 transcripts, and a quadratic (P < 0.001) increase in the activities of GSH-Px and T-AOC in jejunal mucosa. The expression of CYP1A4, CYP2D17 increased and CYP1B1, CYP2A6 decreased linearly (P < 0.001) with dietary curcumin concentrations. In addition, dietary curcumin increased gene expression of GST, MRP6, and ABCB1 in jejunal mucosa. In conclusion, dietary supplementation with 200 to 800 mg/kg curcumin enhanced the accumulation of curcumin and its metabolites in jejunum as well as increasing the antioxidant capacity and detoxification potential, which play major roles in the protection of duck intestines against damage.

Effects of oxidative stress induced by high dosage of dietary iron ingested on intestinal damage and caecal microbiota in Chinese Yellow broilers.

The objective of this trial was to test the effects of oxidative stress induced by a high dosage of dietary iron on intestinal lesion and the microbiological compositions in caecum in Chinese Yellow broilers. A total of 450 1-day-old male chicks were randomly allotted into three groups. Supplemental iron (0, 700 and 1,400 mg/kg) was added to the basal diet resulting in three treatments containing 245, 908 and 1,651 mg/kg Fe (measured value) in diet respectively. Each treatment consisted of six replicate pens with 25 birds per pen. Jejunal enterocyte ultrastructure was observed by transmission electron microscopy. The results showed that a high dosage of dietary iron induced oxidative stress in broilers. Dilated endoplasmic reticulum (ER), autophagosome formation of jejunal enterocytes and decreased villi were caused by this oxidative stress. Compared to the control, concentration of the malondialdehyde (MDA) in jejunal mucosa in the 908 and 1,651 mg/kg Fe groups increased by 180% (p < .01) and 155% respectively (p < .01); activity of copper-zinc superoxide dismutase (Cu/ZnSOD) increased in jejunum (p < .01); and the concentration of plasma reduced glutathione (GSH) decreased by 34.9% (p < .01) in birds fed 1,651 mg/kg Fe. Gene expression of nuclear factor, erythroid-derived 2-like 2 (Nrf2) and zonula occludens-1 (ZO-1), in the higher dietary Fe groups was enhanced (p < .05). Species of microbial flora in caecum increased caused by oxidative stress. The PCR-DGGE (denaturing gradient gel electrophoresis) dendrograms revealed different microbiota (65% similarity coefficient) between the control and iron-supplemented groups (p < .05). These data suggest high dosage of iron supplement in feed diet can induce oxidative stress in Chinese Yellow broilers, and composition of microbiota in the caecum changed. It implied there should be no addition of excess iron when formulating diets in Chinese Yellow broilers.

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein.

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.

[The application of submental island flap in head and neck surgery].

OBJECTIVE: This article discusses the anatomy, surgical procedure,indication, advantages and disadvantages of the submental island flap in reconstruction of the defects after head and neck tumor surgery, based on our clinical experience. METHODS: The submental island flap was transferred with the submental vessels(A/V) as pedicle. In harvesting the flap, the flap was raised commencing from the contralateral side, dissecting all tissues off the mylohyoid muscles. Submandibular triangle dissection was taken until the facial artery and the facial vein. Then the submandibular gland and submandibular nodes were removed and submental vessels were left. This produces a large skin paddle which can be tunnelled to its recipient site. RESULTS: From Aug. 1998 to Oct. 1999, 16 submental island flaps were performed in our department. 15 flaps survived well and 1 flap failed, with a success rate of 15/16 (93.8%). CONCLUSION: The submental island flap is appropriate for primary reconstruction of the defect after various head and neck tumor surgery. It has a long and reliable pedicle, rapid and simple to raise and free from vessel anastomosis. It leaves a well hidden donor site. However, it can't be applied to these cases whose donor site had been radiated before surgery and whose submental and submandibular triangles lymph nodes were positive.

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments.

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.

Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC.

PgtC is one of the three components of the atypical "two-component" pgt regulatory system. To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene. By transport assays, these suppressors were found to confer constitutive pgtP expression. Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21. One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile. Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined. In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well. These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb. In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity. His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression. The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane.

Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase.

The nucleotide sequence of a 7-kilobase segment of the Bacillus subtilis chromosome containing the entire coding regions for the enzymes catalyzing the first three steps of diaminopimelate synthesis as well as dipicolinate synthase has been determined. This group of functionally related genes, termed the dap operon, were arranged in the order orfY, orfX, asd, dapG, and dapA and were bracketed by potential rho-independent transcription terminators. The asd locus could complement the growth defect of Escherichia coli strains with an asd deletion. Disruption of the dapG locus led to the loss of aspartokinase I, with a phenotype similar to that of the temperature-sensitive dapG mutants described earlier (Roten, C. A. H., Brandt, C., and Karamata, D. (1991) J. Gen. Microbiol. 137, 951-962). The amino acid sequences of the deduced products of the asd, dapG, and dapA loci had high degrees of similarity with those of other aspartate semialdehyde dehydrogenases, aspartokinases, and dihydrodipicolinate synthases, respectively. Disruption of orfX had no effect on growth but caused a sporulation defect, characterized by low sporulation frequencies and heat-sensitive spores, which could be cured by supplementation with dipicolinate, similar to the phenotype of mutants defective in spoVF, the putative structural gene for dipicolinate synthase. Two other open reading frames, upstream of spoVF, encoded the 380 COOH-terminal residues of a protein homologous to mitochondrial processing proteases and an 85-residue polypeptide of unknown function. Transcription initiation sites associated with the orfY-orfX-asd-dapG-dapA gene cluster were mapped by primer extension. The results indicate that during vegetative growth, the three distal genes of the dap operon, asd, dapG, and dapA, are transcribed as a unit and orfY and orfX are not expressed, whereas at stage 5 of sporulation two separate transcripts are produced, one comprising all five genes, the other just the three distal genes of the operon.

Genetic evidence for modulation of the activator by two regulatory proteins involved in the exogenous induction of phosphoglycerate transport in Salmonella typhimurium.

Previous work from this laboratory has identified in a fragment of DNA, cloned from Salmonella typhimurium, two genes involved in the exogenous induction of phosphoglycerate transport. These two genes, the transporter gene, pgtP, and the activator gene, pgtA, are closely linked physically; they are only 3.4 kilobases apart. In the accompanying paper, we describe the determination of the nucleotide sequence of this 3.4-kilobase DNA segment and show that this segment contains two genes, pgtB and pgtC, encoding two polypeptides of 593 and 397 amino acid residues, respectively. This paper presents an analysis of the effects of insertions and deletions in pgtBC on the expression of pgtP gene and on the expression of lacZ fused to the pgtP gene. The results indicate that both pgtBC genes are necessary for expression of the pgtP gene. Strikingly, deletion of both genes resulted in a constitutive phenotype, suggesting that PgtB and PgtC polypeptides modulate PgtA activity. The expression of the pgtP gene appears to be regulated by the pgtA gene product, which acts as an activator. A model of induction is proposed in which the central feature is the interaction of the three regulatory proteins in the membrane such that the activity of the activator (PgtA) is subject to modulation by the binding of an inducer.

Nucleotide sequence and transcription start point of the phosphoglycerate transporter gene of Salmonella typhimurium.

We identified the phosphoglycerate transporter gene of Salmonella typhimurium and its polypeptide product and determined the nucleotide sequence of the gene. The predicted translation product was a protein of 406 amino acid residues and was extremely hydrophobic, a feature that is consistent with its role in membrane transport. Hydropathy analysis suggested that there are eight transmembrane segments of at least 20 amino acid residues for the protein. The transcription start point was mapped to lie at position -44 relative to the putative translational initiation start point. Comparison of PgtP with UhpT and GlpT, the membrane-bound proteins involved in the transport of hexose-6-phosphate and glycerol-3-phosphate, respectively, revealed a very high degree of amino acid sequence similarity among them, reflecting not only similar structures and functions among these polypeptides but also a common evolutionary origin for them.