Effects of Glutamine on Lymphocyte Proliferation and Intestinal Mucosal Immune Response in Heat-Stressed Broilers

To investigate the protective effect of glutamine (Gln) on lymphocyte proliferation and the intestinal mucosal immune response in heat-stressed broilers, 360 21-day-old Arbor Acres (AA) broilers were assigned to 4 groups in a completely randomized design, each of which included 6 replicates with 15 birds per replicate for 21 days. The chickens were fed a basal diet under no stress (NS group), a basal diet under heat stress (HT group), or a basal diet under heat stress with the addition of either 0.5 % or 1.0 % Gln. The results showed that the broilers in the HT group exhibited fewer proliferating peripheral lymphocytes, a lower growth performance, phagocytic rate and index of neutrophils, fewer goblet cells in whole intestine and intraepithelial lymphocyte (IEL) cells in the ileum, a lower sIgA content in the duodenum and the jejunum, a lower immunoglobulin content of serum and intestinal mucosa, than those of the NS group (p<0.05). Diets supplemented with Gln increased growth performance, the number of proliferating peripheral lymphocytes, the phagocytic rate and phagocytic index of neutrophils, the number of whole intestine goblet cells and ileum IEL cells, the sIgA contents of the duodenum and the jejunum, and the immunoglobulin contents of serum and intestinal mucosa (p<0.05) in broilers exposed to HT. In conclusion, Gln can enhance intestinal immune function in broiler chickens by stimulating T and B lymphocyte proliferation, increasing the number of goblet cells and IEL cells, as well as increasing the content of sIgA and immunoglobulin secretion.

Effects of Glutamine on Lymphocyte Proliferation and Intestinal Mucosal Immune Response in Heat-Stressed Broilers

To investigate the protective effect of glutamine (Gln) on lymphocyte proliferation and the intestinal mucosal immune response in heat-stressed broilers, 360 21-day-old Arbor Acres (AA) broilers were assigned to 4 groups in a completely randomized design, each of which included 6 replicates with 15 birds per replicate for 21 days. The chickens were fed a basal diet under no stress (NS group), a basal diet under heat stress (HT group), or a basal diet under heat stress with the addition of either 0.5 % or 1.0 % Gln. The results showed that the broilers in the HT group exhibited fewer proliferating peripheral lymphocytes, a lower growth performance, phagocytic rate and index of neutrophils, fewer goblet cells in whole intestine and intraepithelial lymphocyte (IEL) cells in the ileum, a lower sIgA content in the duodenum and the jejunum, a lower immunoglobulin content of serum and intestinal mucosa, than those of the NS group (p<0.05). Diets supplemented with Gln increased growth performance, the number of proliferating peripheral lymphocytes, the phagocytic rate and phagocytic index of neutrophils, the number of whole intestine goblet cells and ileum IEL cells, the sIgA contents of the duodenum and the jejunum, and the immunoglobulin contents of serum and intestinal mucosa (p<0.05) in broilers exposed to HT. In conclusion, Gln can enhance intestinal immune function in broiler chickens by stimulating T and B lymphocyte proliferation, increasing the number of goblet cells and IEL cells, as well as increasing the content of sIgA and immunoglobulin secretion.(AU)

Effects of Glutamine on the Mucosal Structure and Immune Cells in the Intestines of Broiler Chickens Challenged with Salmonella Enteritidis

The aim of this study was to investigate the effects of glutamine (Gln) on the intestinal mucosal structure and immune cells of broilers infected with Salmonella Enteritidis. 160 1-d-old commercial Arbor Acres (AA) broilers were randomly selected to receive one of four treatments, each of which had 5 replicates. Each replicate consisted of 8 chicks subjected to a 21-d feeding trial. Group I served as the unchallenged (CON). All birds in groups II (SCC) - IV were challenged with 2.0 × 104 CFU/mL of S. Enteritidis. The birds in groups III and IV were treated with 0.5% and 1.0% Gln. The results showed that S. Enteritidis infection led to a decrease in the relative length and weight, villus height:crypt depth (VH:CD) of the jejunum and ileum, the number of intraepithelial lymphocyte cells, and number of goblet cells and an increase in the number of mast goblet cells compared with the measurements of these parameters in the CON group (p 0.05). In addition, the Gln groups had increased relative length and weight, VH:CD of the jejunum and ileum, numbers of intraepithelial lymphocyte cells, and numbers of goblet cells and decreased crypt depth in the jejunum and ileum and numbers of mast goblet cells compared with the measurements of these parameters in the SCC group (p 0.05). It was concluded that Gln added to broiler diets can effectively alleviate the intestinal mucosal damage caused by S. Enteritidis infection and improve its normal defense barrier function.

Effects of Glutamine on the Mucosal Structure and Immune Cells in the Intestines of Broiler Chickens Challenged with Salmonella Enteritidis

The aim of this study was to investigate the effects of glutamine (Gln) on the intestinal mucosal structure and immune cells of broilers infected with Salmonella Enteritidis. 160 1-d-old commercial Arbor Acres (AA) broilers were randomly selected to receive one of four treatments, each of which had 5 replicates. Each replicate consisted of 8 chicks subjected to a 21-d feeding trial. Group I served as the unchallenged (CON). All birds in groups II (SCC) - IV were challenged with 2.0 × 104 CFU/mL of S. Enteritidis. The birds in groups III and IV were treated with 0.5% and 1.0% Gln. The results showed that S. Enteritidis infection led to a decrease in the relative length and weight, villus height:crypt depth (VH:CD) of the jejunum and ileum, the number of intraepithelial lymphocyte cells, and number of goblet cells and an increase in the number of mast goblet cells compared with the measurements of these parameters in the CON group (p 0.05). In addition, the Gln groups had increased relative length and weight, VH:CD of the jejunum and ileum, numbers of intraepithelial lymphocyte cells, and numbers of goblet cells and decreased crypt depth in the jejunum and ileum and numbers of mast goblet cells compared with the measurements of these parameters in the SCC group (p 0.05). It was concluded that Gln added to broiler diets can effectively alleviate the intestinal mucosal damage caused by S. Enteritidis infection and improve its normal defense barrier function.(AU)

Meta-analysis of the IL-10 promoter polymorphisms and pediatric asthma susceptibility.

The results of previous epidemiological studies exploring the relationship between interleukin-10 (IL-10) promoter polymorphisms and susceptibility to pediatric asthma are not consistent. Therefore, we have performed a systematic review and meta-analysis to provide a more convincing conclusion. Odds ratios (OR) with their 95% confidence intervals (CIs) were used to evaluate the strength of association between the IL-10 promoter polymorphisms and susceptibility to pediatric asthma. Publication bias was examined by Begg's funnel plots and the Egger test. A detailed literature search based on stringent parameters yielded sixteen relevant studies, comprising 2494 cases and 2160 controls. The overall population showed no significant association between the IL-10 -1082G/A polymorphism and pediatric asthma risk in any of the genetic models (dominant model: OR = 1.583, 95%CI = 0.614-4.076, P = 0.342; allelic model: OR = 1.214, 95%CI = 0.748-1.971, P = 0.433; additive model: OR = 2.240, 95%CI = 0.950-5.277, P = 0.065; recessive model: OR = 1.435, 95%CI = 0.659-3.128, P = 0.363). Subgroup analyses revealed a significant association between different ethnicity and atopic status subgroups. However, there was no evidence of a significant association between the other two polymorphisms (-819C/ T and -592C/A) and pediatric asthma in our study. No significant publication bias was observed in this meta-analysis. The results of this study indicate that the IL-10 -1082G/A polymorphism might be a risk factor for asthma in children. However, because of the small sample size included in the subgroup analyses, the results should be interpreted with caution.

Development of microsatellite loci for Taxus wallichiana var. wallichiana (Taxaceae) and cross-amplification in Taxaceae.

Nine polymorphic microsatellite loci were isolated and characterized in Taxus wallichiana var. wallichiana, an endangered species in China. The number of alleles per locus ranged from 2 to 20. Observed and expected heterozygosities varied from 0.0260 to 0.5325 and 0.3603 to 0.9231, respectively. Positive cross-amplification of the 9 loci was observed in 2 other varieties of T. wallichiana and 4 other Taxaceae species. These loci will be of value for studying population genetic structures and for genetic resource conservation in T. wallichiana and other Taxus species.

Genetic diversity analysis of Capparis spinosa L. populations by using ISSR markers.

Capparis spinosa L. is an important medicinal species in the Xinjiang Province of China. Ten natural populations of C. spinosa from 3 locations in North, Central, and South Xinjiang were studied using morphological trait inter simple sequence repeat (ISSR) molecular markers to assess the genetic diversity and population structure. In this study, the 10 ISSR primers produced 313 amplified DNA fragments, with 52% of fragments being polymorphic. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis indicated that 10 C. spinosa populations were clustered into 3 geographically distinct groups. The Nei gene of C. spinosa populations in different regions had Diversity and Shannon's information index ranges of 0.1312-0.2001 and 0.1004-0.1875, respectively. The 362 markers were used to construct the dendrogram based on the UPGMA cluster analysis. The dendrogram indicated that 10 populations of C. spinosa were clustered into 3 geographically distinct groups. The results showed these genotypes have high genetic diversity, and can be used for an alternative breeding program.

Effect of temperature on endogenous hormone levels and opposite phyllotaxy in maize leaf primordial.

Newly identified maize (Zea mays) mutants with opposite leaf phyllotaxy are important in the study of the maize crop. Previous studies have revealed the developmental mechanism of opposite phyllotaxy on the physiological, cellular, and molecular levels. However, there have been few reports regarding the effects of changes in endogenous hormone levels in maize leaf primordia under different conditions. We conducted field studies to examine the influence of different environmental factors on leaf primordia differentiation. Our results indicated that compared with other major environmental factors, temperature was significantly positively correlated with the ratio of maize plants with opposite phyllotaxy. We examined endogenous hormone levels in maize at different temperatures using an enzyme-linked immunosorbent assay. The results showed that the ratio of maize plants with opposite phyllotaxy was mainly influenced by the cytokinin/auxin ratio. In addition, at the same temperature, the ratio of cytokinin/auxin in maize with opposite phyllotaxy was significantly higher than that near isogenic lines with alternate phyllotaxy.

Cloning and functional analysis of the promoter of a maize starch synthase III gene (ZmDULL1).

The ZmDULL1 gene encodes a starch synthase and is a determinant of the structure of endosperm starch in maize (Zea mays L.). However, little is known regarding the regulatory mechanism of the ZmDULL1 gene. In this study, we isolated and characterized the ZmDULL1 promoter (PDULL1), which is the 5' flanking region of ZmDULL1 in maize. Sequence analysis showed that several cis-acting elements important for endosperm expression (GCN4_motif and AACA-element) were located within the promoter. A series of PDULL1 deletion derivatives, PDULL1-1-PDULL1-4, from the translation start code (-1676, -1216, -740, and -343) were fused to the ß-glucuronidase (GUS) reporter gene. Each deletion construct was transformed into rice using the Agrobacterium-mediated method, and then GUS activity was measured in transgenic plants. The results showed that PDULL1 was an endosperm-specific promoter. Further analysis showed that the promoter sequence (-343 to -1 base pairs) was sufficient for mediating GUS gene expression in endosperm. These results indicate that the region from -343 to -1 base pairs of PDULL1 is valuable for transgenic rice breeding and genetic engineering studies.

Systematic meta-analysis of the association between monocyte chemoattractant protein-1 -2518A/G polymorphism and risk of tuberculosis.

Numerous studies have been conducted to investigate the association between 2518A/G polymorphisms in the monocyte chemoattractant protein-1 (MCP-1) gene and the risk of tuberculosis (TB). However, the results have been inconsistent and inconclusive. In this study, we performed a meta-analysis to evaluate the association between the MCP-1 -2518A/G polymorphism and TB. The National Center for Biotechnology Information Global Cross Database and Google Scholar database were searched for relative studies. A total of 22 case-control studies that included 7332 cases and 8004 controls for the -2518A/G single-nucleotide polymorphism were identified. The results revealed an association between the MCP-1 -2518A/G polymorphism and human TB susceptibility under a recessive model (GG vs GA+AA), dominant model (GG+GA vs AA), and homozygote comparison (GG vs AA) model for the entire database. For the dominant model, the overall odds ratio was 1.34 (95% confidence interval, 1.10-1.64, P = 0.004). For the recessive model, the overall odds ratio was 1.46 (95% confidence interval, 1.15- 1.86, P = 0.002). For the homozygote comparison, the overall odds ratio was 1.67 (95% confidence interval, 1.20-2.32, P = 0.002). In the subgroup analysis by ethnicity, significantly elevated risks were found in Asians and Americans, but not in Africans and Europeans. We also used the Begg and Egger tests to examine publication bias, and no major publication bias was detected. Our results indicate that there is an association between the MCP-1 -2518A/G polymorphism and human TB susceptibility.

Screening relevant genes of tolerance to low phosphorus in maize using cDNA-amplified fragment length polymorphism.

Soil contains a large amount of phosphorus, but plants cannot absorb most of this phosphorus effectively. Low inorganic phosphorus has been singled out as a major constraint that leads to a perpetually low Zea mays (maize) grain yield. The fundamental approach to solving this problem is to screen new genes of low phosphorous (LP) tolerance. Consequently, the exploration and utilization of LP-tolerant genes are of great significance in plants. The maize inbred line 178 is an inbred LP-tolerant line. In the current study, the expression of this inbred line was induced under the stress of LP conditions. We applied cDNA-amplified fragment length polymorphism to screen LP-tolerant genes and obtained and sequenced 78 differentially expressed gene fragments. Their functions were predicted via bioinformatic analysis. There were no function annotations for 8 differentially expressed fragments. Nine genes exhibited high homology to Arabidopsis thaliana and Oryza sativa genes involved in phosphorus metabolism. This study lays a good foundation for further cloning and verification of the genes involved in phosphorus metabolism in maize.

Association of the IL-4R Q576R polymorphism and asthma in the Chinese Han population: a meta-analysis.

The IL-4R Q576R polymorphism has been reported to increase susceptibility to asthma, but the results are controversial. Thus, we performed a meta-analysis to evaluate the association of the IL-4R Q576R polymorphism and asthma risk in the Chinese Han population. A total of sixteen eligible case-control studies that evaluated the relationship between the IL-4R Q576R polymorphism and asthma in the Chinese Han population were obtained by comprehensive literature search incorporating electronic databases, and included 2077 asthma cases and 1589 controls. Our analysis detected a significant association between the IL-4R Q576R polymorphism and the risk of asthma in the Chinese Han population (Allelic model: OR = 1.481, 95%CI = 1.134-1.935, P = 0.004; Dominant model: OR = 1.542, 95%CI = 1.194-1.990, P = 0.001; Recessive model: OR = 1.695, 95%CI = 1.170-2.456, P = 0.005, Additive model: OR = 1.897, 95%CI = 1.299-2.771, P = 0.005). The year of publication and size of total sample might be sources of between-study heterogeneity. Upon subgroup analysis by size of total sample of each study, the significant association only remained in a subgroup with a small sample size. In summary, our meta-analysis suggested that the IL-4R Q576R polymorphism is associated with asthma in the Chinese Han population.

Overexpression of an endo-1,4-ß-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion.

Wnt/ß-catenin aids in regulating the proliferation of hepG2 cells mediated by thy-1.

Cancer stem cells have been found to play important roles in carcinoma. Although thy-1 has been identified as a potential stem cell marker of liver cancer, whether the Wnt/ß-catenin signaling pathway plays an important role in regulating hepatocarcinoma proliferation and apoptosis mediated by thy-1 remains unknown. Our results showed that high thy-1 expression caused hepG2 cells transfected with a pReceiver-M29/thy-1 eukaryotic expression vector to exhibit obvious heteromorphism, featuring double or multiple nuclei and weaker apoptosis. A high expression of ß-catenin, as a critical signaling protein of Wnt/ß-catenin, and its downstream transcription factor, cyclinD1, were detected in transfected hepG2 cells. We also used aspirin as an inhibitor of the Wnt signaling pathway in the treatment of hepG2 cells transfected with the pReceiver-M29/thy-1 expression vector to make detailed observations of apoptosis in hepG2 cells as well as the differential expression of ß-catenin, cyclinD1, and thy-1. An increasing apoptosis rate was detected in the hepG2 cells and downregulated expression of the three proteins was detected. Hence, we suggest that thy-1 upregulation promotes the proliferation and inhibits apoptosis of hepG2 cells, and that these processes are regulated by the Wnt/ß- catenin signaling pathway.

A genome-wide analysis of the ERF gene family in sorghum.

The ethylene response factor (ERF) family are members of the APETALA2 (AP2)/ERF transcription factor superfamily; they are known to play an important role in plant adaptation to biotic and abiotic stress. ERF genes have been studied in Arabidopsis, rice, grape, and maize; however, there are few reports of ERF genes in sorghum. We identified 105 sorghum ERF (SbERF) genes, which were categorized into 12 groups (A-1 to A-6 and B-1 to B-6) based on their sequence similarity, and this new method of classification for ERF genes was then further characterized. A comprehensive bioinformatic analysis of SbERF genes was performed using a sorghum genomic database, to analyze the phylogeny of SbERF genes, identify other conserved motifs apart from the AP2/ERF domain, map SbERF genes to the 10 sorghum chromosomes, and determine the tissue-specific expression patterns of SbERF genes. Gene clustering indicates that SbERF genes were generated by tandem duplications. Comparison of SbERF genes with maize ERF homologs suggests lateral gene transfer between monocot species. These results can contribute to our understanting of the evolution of the ERF gene family.

RNA interference-mediated silencing of the starch branching enzyme gene improves amylose content in rice.

Amylose and amylopectin are the 2 major components of plant storage starch. The rice starch branching enzyme (RBE) plays an important role in the starch components of rice. In the present study, we selected a specific 195-bp segment from the RBE3 gene to construct hairpin DNA, which was driven by an endosperm-specific high molecular weight glutenin promoter to regulate the biosynthesis of starch. An RNA interference plasmid for the RBE3 gene was constructed to form double-stranded RNA. Following Agrobacterium-mediated rice transformation (in the cultivar Zhonghua 11), 41 transgenic plants were identified using PCR and Southern blot analysis. Semi-quantitative real-time PCR revealed that RBE3 gene expression was significantly reduced in immature transgenic seeds. Transgenic rice amylose content had an average increase of 140%. The highest rice amylose content was 47.61% and the growth rate increased 238% compared to the non-transgenic controls. Branching enzyme II activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight in transgenic plants. The present study indicated that amylose content was negatively correlated with branching enzyme II activity, spike size, and thousand-grain weight.

Genome-wide analysis of immunophilin FKBP genes and expression patterns in Zea mays.

The receptors for the immunosuppression drugs FK506 and rapamycin are called FKBPs (FK506-binding proteins). FKBPs comprise a large family; they are found in many species, including bacteria, fungi, animals, and plants. As a class of peptidyl-prolyl cis-trans isomerase enzymes, the FKBP genes have been the focus of recent studies on plant stress tolerance and immunology. We identified and analyzed gene families encoding these proteins in maize using computational and molecular biology approaches. Thirty genes were found to encode putative FKBPs according to their FK506-binding domain. The FKBP genes can be classified into single domain and multiple domain members based on the number of the domains. By analysis of the physical locations, the 30 FKBP genes were found to be widely distributed on 10 chromosomes. After analysis of the FKBP phylogenetic tree in the maize genome, we found that the 30 genes revealed two major clades. Gene duplication played a major role in the evolution of FKBP genes, which suggests that the FKBP genes in maize have a pattern significantly different from that of these genes in rice. Based on semi-quantitative RT-PCR, we found that the 30 FKBPs were expressed differently in various tissues in maize, which suggests that FKBP genes play different roles in each tissue. Several FKBPs were expressed at higher levels in roots, indicating that these genes in maize may have similar or overlapping functions.

Molecular mapping of genes for opposite leafing in maize using simple-sequence repeat markers.

Maize with opposite phyllotaxy (OP) and also initiating ears in opposite pairs is an aberrant mutant and also precious material for maize breeding and plant evolution studies. Mapping and identifying the markers closely linked to genes for the OP trait are essential for cloning the gene and marker-assisted selection in breeding. We established H14D, a near-isogenic line of the OP trait with H53 genetic background. We found that the OP trait is regulated by two independent dominant genes with mutually complementary relations, named Opp-1 and Opp-2. Screening of seven simple-sequence repeat (SSR) markers among the 105 pairs of SSR primers showed polymorphism between the inbred lines H14D and H53. The polymorphic SSR markers were then used to determine linkage with the trait in an F(2) population with 441 progeny, suggesting that SSR marker umc2094 in the Bin2.01 region is linked with Opp-1 at 6.7 cM, and bnlg1831 in Bin2.06 is linked with Opp-2 at 6.1 cM. Further investigation showed that bnlg1092 and umc1028 are linked to Opp-1 and Opp-2 genes, with genetic distances of 12.2 and 1.9 cM. It was also found that the four SSR markers flank the two OP genes, respectively. These results will be useful for marker-assisted selection breeding of OP maize and will also strengthen the basis for cloning of the opposite leafing gene.

Genome-wide identification, classification, and analysis of two-component signal system genes in maize.

Cytokinins play many vital roles in plant development and physiology. In plants, cytokinin signals are sensed and transduced by the two-component signal system. This signaling cascade is typically composed of three proteins: a sensory histidine kinase, a histidine phosphotransfer protein, and a response regulator. Through a comprehensive genome-wide analysis of the maize (Zea mays) genome, 48 genes were identified, including 11 ZmHKs, 9 ZmHPs, and 28 ZmRRs (21 A-type ZmRRs and 7 B-type ZmRRs). Using maize genome sequence databases, we analyzed conserved protein motifs and established phylogenetic relationships based on gene structure, homology, and chromosomal location. The duplication of these two-component system genes in the maize genome corresponded to the clusters of these genes in the phylogenetic trees. Sequence analysis of the duplicate genes demonstrated that one gene may be in gene duplication, and that there was significant variation in the evolutionary history of the different gene families. We assessed the expression levels of all ZmRRs in the leaves and roots by reverse transcription PCR; they were all found to be active. Our results provide a foundation for functional and evolutionary studies on maize two-component signal system proteins.

Molecular and functional analysis of the poly-ß-hydroxybutyrate biosynthesis operon of Pseudomonas sp BJ-1.

The operon comprising the genes for poly-ß-hydroxybutyrate (PHB) biosynthesis in Pseudomonas sp BJ-1 was cloned and sequenced. Sequence analysis of 8991 bp revealed that the regions contain two related operons. The first operon contains the three genes phbA, phbB and phbC, and the other contains the two genes flp1 and flp2. The deduced amino acid sequences of PHBA and PHBB showed high identity with other bacterial PHB genes. Transcription of the three genes of the first operon is controlled by a single hypothetical promoter region, whereas the other two flp genes are controlled by two hypothetical promoter regions. Analysis of expressed protein at different times showed that PHBA protein levels increased from 0 to 4 h; PHBB and PHBC showed similar kinetics. Detection of enzyme activity showed three proteins with bioactivity and biological function in the synthesis of PHB intermediates.