太平洋牡蛎i型溶菌酶基因的克隆与重组高效表达

在双壳类软体动物牡蛎体内,溶菌酶(Lysozyme)在实现宿主免疫防御,破坏和消除侵入体内的病原中发挥着重要的作用.根据GenBank已有的太平洋牡蛎溶菌酶的全长cDNA序列(GenBank:AB179775),通过RT-PCR技术,从太平洋牡蛎(Crassostrea gigas)中克隆得到溶菌酶(简称为CgLys)基因的开放阅读框(open reading frame,ORF)序列.生物信息软件分析表明,其ORF为414 bp,编码137个氨基酸(aa),前20个aa为信号肽,成熟肽由117个aa组成,其分子量为13.2 kD.通过构建分子系统发育树对其同源性进行分析比较,初步推断该CgLys属于i型溶菌酶.将该CgLys基因的成熟肽亚克隆进原核表达载体pET-32a(+)中,构建重组质粒pET32a(+)-CgLys,再转化至大肠杆菌BL21(DE3)pLysS.该基因工程菌经IPTG诱导发酵后,成功高效地表达了重组CgLys蛋白,其分子质量约为18 kD.该重组CgLya蛋白主要存在于细菌裂解液的上清液中,即以可溶性蛋白形式存在.上述结果将显著简化后续的蛋白纯化过程,为今后扩大规模生产牡蛎溶菌酶提供参考.     

The influence of hydrocortisone on cellular defence mechanisms of Biomphalaria glabrata

Since the internal defense system of mollusks consists of cellular and humoral mechanisms, we examined the role of hydrocortisone in mollusks defense cells and the influence of this steroid on the development of Schistosoma mansoni in its intermediary host. Hydrocortisone had an immunosuppressive action in Biomphalaria glabrata, as reflected in the reduced number of defense cells and the altered cell physiology. Histopathological analysis showed that hydrocortisone facilitated the intramolluscan development of S. mansoni, by reducing the extent of the inflammatory response, seen as a greater number of viable sporocysts with no surrounding hemocytes.     

Oxidative stress and expression of chaperones in aging mollusks

The mechanisms of aging are not well understood in animals with continuous growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. We studied the effects of age on oxidative stress, cellular defense mechanisms (including two major antioxidant enzymes, superoxide dismutase (SOD) and catalase), and molecular chaperones in two mollusks--eastern oysters Crassostrea virginica and hard clams Mercenaria mercenaria. In order to detect the age-related changes in these parameters, correction for the effects of size was performed where appropriate to account for growth-related dilution. Fluorescent age pigments accumulated with age in both species. Protein carbonyls did not change with age or size indicating that they are not a good marker of aging in mollusks possibly due to the fast turnover and degradation of oxidized proteins in growing tissues. SOD did not show a compensatory increase with aging in either species, while catalase significantly decreased with age. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age-related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species-specific. HSP70 increased and HSP90 declined with age in clams, whereas in oysters HSP70 expression did not change, and HSP90 increased with aging.     

Genes expressed in Ascochyta rabiei-inoculated chickpea plants and elicited cell cultures as detected by differential cDNA-hybridization

In response to the exogenous application of elicitors and attempted invasion by pathogens, plants exhibit a wide range of defense reactions. To understand the defense mechanisms at the level of gene activation and deactivation, differential screenings were performed to isolate cDNA clones which are differentially expressed in pathogen-inoculated resistant chickpea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arranged in 5 groups, namely defense-related pathways, signal transduction pathways, regulation of gene expression, catabolic pathways and primary metabolism. Most of these genes were activated although several genes were also found to be suppressed. We discuss the plausible functions of cDNA products in plant defense responses. The cDNAs provide a variety of tools to investigate molecular mechanisms of defense responses and clearly reflect the massive genomic and metabolic changes which occur during manifestation of antimicrobial defense.     

Differences in the number of hemocytes in the snail host Biomphalaria tenagophila, resistant and susceptible to Schistosoma mansoni infection

The relationships between schistosomiasis and its intermediate host, mollusks of the genus Biomphalaria, have been a concern for decades. It is known that the vector mollusk shows different susceptibility against parasite infection, whose occurrence depends on the interaction between the forms of trematode larvae and the host defense cells. These cells are called amebocytes or hemocytes and are responsible for the recognition of foreign bodies and for phagocytosis and cytotoxic reactions. The defense cells mediate the modulation of the resistant and susceptible phenotypes of the mollusk. Two main types of hemocytes are found in the Biomphalaria hemolymph: the granulocytes and the hyalinocytes. We studied the variation in the number (kinetics) of hemocytes for 24 h after exposing the parasite to genetically selected and non-selected strains of Biomphalaria tenagophila, susceptible or not to infection by Schistosoma mansoni. The differences were analyzed referred to the variations in the number of hemocytes in mollusks susceptible or not to infection by S. mansoni. The hemolymph of the selected and non-selected snails was collected, and hemocytes were counted using a Neubauer chamber at six designated periods: 0 h (control, non-exposed individuals), 2 h, 6 h, 12 h, 18 h and, 24 h after parasite exposure. Samples of hemolymph of five selected mollusks and five non-selected mollusks were separately used at each counting time. There was a significant variation in the number of hemocytes between the strains, which indicates that defense cells have different behaviors in resistant and susceptible mollusks.     

Host defense mechanisms of cephalopods

Humoral and cellular mechanisms of defense have been described for cephalopods, a relatively advanced group of mollusks. Typical of other mollusks, cephalopod agglutinins are the most documented component of humoral immunity. Lectins, which have agglutinating properties, have been described and characterized from octopuses. Agglutinins from cephalopod hemolymph have also been shown to agglutinate a variety of vertebrate red blood cells, as well as potential bacterial pathogens. Hemocytes are the primary component of cellular immunity. Although the hemocyte role in phagocytosis has been extensively studied in other mollusks, the mechanisms of phagocytosis have not been described extensively for cephalopods. Cephalopod hemocytes have phagocytic capabilities and may function in encapsulation and neutralization of foreign substances; however, the effects of environmental factors and the full extent of phagocytic capabilities of cephalopod hemocytes have not been reported. Hemocytes from cephalopods have a role in wound healing and inflammation which have been reported in detail by several investigators.     

New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.     

Purification and Characterization of Trochus radiatus Derived Low Molecular Weight Bactericidal Polypeptide Active Against ESKAPE Pathogens

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides comprise core components of innate defense and act as first-line defense molecules in most marine mollusks....     

Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica

We generated an acetylcholine-binding protein from Aplysia californica by synthesis of a cDNA found in existing data bases and its expression in mammalian cell culture. Its subunit assembly and ligand recognition behavior were compared with the binding protein previously derived from Lymnaea stagnalis. The secreted proteins were purified by elution from columns of attached antibodies directed to the FLAG epitope encoded in the expression construct. Although the sequences of the two proteins from marine and fresh water mollusks exhibit the characteristic features of the extracellular domain of the nicotinic receptor, they only possess 33% amino acid identity. Both assemble as stable pentamers with five binding sites per pentamer, yet they show distinguishing features of stability and sensitivity to epitope tag placement. Both proteins exhibit changes in tryptophan fluorescence upon ligand binding; however, the magnitude of the changes differs greatly. Moreover, certain ligands show marked differences in dissociation constants for the two proteins and can be regarded as distinguishing or signature ligands. Hence, the two soluble proteins from mollusks, which can be studied by a variety of physical methods, become discrete surrogate proteins for the extracellular domains of distinct subtypes of nicotinic acetylcholine receptors.     

Early development of the Japanese spiny oyster (Saccostrea kegaki): characterization of some genetic markers

The phylum Mollusca is one of the major groups of Lophotrochozoa. Although mollusks exhibit great morphological diversity, only a few comparative embryological studies have been performed on this group. In the present study, to begin understanding the molecular development of the diverse morphology among mollusks, we observed early embryogenesis in a bivalve, the Japanese spiny oyster, Saccostrea kegaki. Although several studies have begun to reveal the genetic machinery for early development in gastropods, very little molecular information is available on bivalve embryogenesis. Thus, as a step toward identifying tissue-specific gene markers, we sequenced about 100 cDNA clones picked randomly from a gastrula-stage cDNA library. This basic information on bivalve embryology will be useful for further studies on the development and evolution of mollusks.