Cloning,expression and cellular localization of the Doublesex gene in the water flea, Daphnia carinata, during different developmental stages

In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata, designated DapcaDsx1, was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Antenna-predominant and male-biased CSP19 of Sesamia inferens is able to bind the female sex pheromones and host plant volatiles

Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni–ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12–8.75 μM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49–1.78 μM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles.     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

Development of quantitative PCR and metagenomics-based approaches for strain quantification of a defined mixed-strain starter culture

Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited.     

金鱼藤中ApNAP基因的克隆和表达模式分析

金鱼藤(Asarina procumbens)是玄参科金鱼藤属多年生藤本植物,在园林中常用于垂直绿化,是良好的观赏植物,但金鱼藤叶片衰老后很快干枯并脱落,仅留光秃的茎蔓,使其观赏价值大大降低。AtNAP是拟南芥NAC基因家族中与衰老相关的转录因子,在调控叶片衰老过程中有重要的作用。本研究以金鱼藤衰老叶片为材料,运用RT-PCR和RACE技术克隆金鱼藤中ApNAP基因cDNA序列。结果显示,该基因位于细胞核中,cDNA全长为1341 bp,开放阅读框为936 bp,编码311个氨基酸,相对分子量为35.21 kD,等电点为9.13;ApNAP基因组全长1526 bp,含有2个内含子和3个外显子。多重序列比对表明,ApNAP属于NAC转录因子家族。系统进化树分析显示,ApNAP属于NAP亚组,与AtNAM的同源关系最近。实时荧光定量PCR结果表明,受自然状态下衰老和黑暗处理的诱导,ApNAP基因表达量增加,这进一步说明ApNAP基因参与调控金鱼藤叶片衰老的过程。该基因的分离对利用转基因技术延缓金鱼藤叶片衰老,提高金鱼藤在园林绿化中的观赏价值具有实际意义。     

Identification,characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base‐excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full‐length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis‐element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub‐functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.