Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation

Bachère, Evelyne; Chagot, Dominique; Grizel, Henri

doi:10.1016/0145-305x(88)90071-7

Cited by 140 publications

(60 citation statements)

References 20 publications

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“…As previously described (Bachere et al 1988), this difficulty may be greatly reduced by instantaneously diluting the hemolymph, during withdrawal, in MAS, a n efficient anti-aggregant. In this way, it is subsequently possible to aliquot hemolymph into several samples which are strictly homogeneous, quantitatively and qualitatively, and consequently well- …”

Section: Discussionmentioning

confidence: 93%

“…In order to understand the influence of hemogram characteristics on CL responses, the respective involvement of each cell type in CL activity can best be studied with pure hemocyte populations separated using isopycnic and elutriation centrifugation (Bachere et al 1988).…”

Section: Discussionmentioning

confidence: 99%

“…This paper describes the establishment of experi- (Bachere et al 1988). The hemolymph samples were used either separately or pooled (10 individual hemolymphs).…”

Section: Introductionmentioning

confidence: 99%

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Lumhol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas

Bachère¹,

Hervio²,

Mialhe³

1991

Dis. Aquat. Org.

107

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Microbicidal phagocytic function and oxidative metabolism of the hemocytes of 2 oyster species, Ostrea edulis and Crassostrea gigas, were investigated using a luminol-enhanced chemiluminescence (CL) technique. First, experimental parameters adapted to marine bivalve hemocytes were established on hemolymph pools of C. gigas in order to obtain qualitatively and quantitatively homogeneous samples and so to perform statistically comparable assays. The use of Modified Alsever Solution (MAS) allowed hemocytes to b e kept non-aggregated and in a non-stimulated state until starting the assays. A number of aliquots of 2 X lo5 hemocytes with a n MAS final concentration of 3.5 % showed high chemiluminescent response after stimulation by zymosan particles A partic1e:hemocyte ratio of 80:1 gave optimal CL activity. Activity was inhibited by cytochalasin B, a phagocyt~c inhibitor, and by sodlum azide, the latter indicating the involvement of oxygen products. Having thus defined suitable parameter values, the CL protocol was then applled to 0. edulis for qualitative a n d quantitative analyses of respiratory burst capacity at the species and individual levels. Results suggested inter-and intra-specific variability of CL responses. Under the same experimental conditions, 0. eduhs hemocytes generally displayed higher CL activities than C. gigas. Moreover, clear individual vanabllity was demonstrated. The same experimental number of hemocytes showed great differences in CL responses between individuals suggesting a posslble correlation with hemogram characteristics such a s cell type percentages.

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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Lumhol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas

Bachère¹,

Hervio²,

Mialhe³

1991

Dis. Aquat. Org.

107

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“…Oysters were challenged by adding heat-killed bacteria Micrococcus luteus, Vibrio splendidus, and Vibrio anguillarum (5 ϫ 10 8 bacteria per liter) in sea-water tanks. Hemolymph was collected at different times (0, 15, 24, and 48 h) in antiaggregant modified Alsever solution (40). Hemocytes were collected by centrifugation (700 ϫ g, 10 min, 4°C).…”

Section: Methodsmentioning

confidence: 99%

Evidence of a bactericidal permeability increasing protein in an invertebrate, theCrassostrea gigas Cg-BPI

González-Aravena

Gueguen

Destoumieux-Garzón

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

125

124

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A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oyster Crassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity against Escherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein. By in situ hybridization, the expression of the C. gigas BPI (Cg-bpi) was found to be induced in hemocytes after oyster bacterial challenge and to be constitutive in various epithelia of unchallenged oysters. Thus, Cg-bpi transcripts were detected in the epithelial cells of tissues/organs in contact with the external environment (mantle, gills, digestive tract, digestive gland diverticula, and gonad follicles). Therefore, Cg-BPI, whose expression profile and biological properties are reminiscent of mammalian BPIs, may provide a first line of defense against potential bacterial invasion. To our knowledge, this is the first characterization of a BPI in an invertebrate.antimicrobial ͉ epithelia ͉ hemocyte ͉ mollusk ͉ oyster innate immunity M arine invertebrates including bivalve mollusks have evolved in the continuous presence of microorganisms. The oysters, such as Crassostrea gigas, harbor a diverse microflora both on their surface (epibiosis) and inside the body cavities and hemolymph (endobiosis). They have developed an efficient immune system for maintaining balance with commensal and pathogenic bacteria, in particular with the Gram-negative Vibrio spp. abundant in their tissues/organs. LPS from Gram-negative bacteria play an important role in the interaction and activation of the innate immune system including the antimicrobial defense (1, 2). In invertebrates, LPSbinding proteins (LBP) participate in the transduction of cellular signals from LPS. LBPs have been characterized in the freshwater crayfish Pacifastacus leniusculus (3), the shrimp Litopenaeus stylirostris (4), the earthworm Eisenia foetida (5), and the silkworm Bombyx mori (6). In mammals, LBP is an acute phase plasma protein constitutively secreted by liver that induces cellular responses (7). In particular, LBP participates in the acute mobilization of circulating neutrophils to sites of tissue injury. Stored in the mobilized neutrophils, antimicrobial peptides and the bactericidal/ permeability-increasing protein (BPI) contribute to the elimination of bacteria (8, 9). BPI, another LBP, is a 55-kDa cationic protein specifically active against Gram-negative bacteria. It increases the permeability of the bacterial membranes (10). Accumulated extracellularly, BPI opsonizes bacteria, which enhances phagocytosis by neutrophils (11). LBP and BPI are structurally related, with 45% sequence identity. They have a coordinated function in the response to invading bacteria. The antibacterial BPI displays LPSneutralizing properties and suppresses LPS inflammatory activity whereas LBP is an acute-phase reactant (8, 12) that displays a concen...

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“…The shrimp were acclimated in the aerated seawater at 25±1°C for 5-7 days in the lab before the experiment. The hemolymph was withdrawn from the ventral sinus located at the first abdominal segment using an equal volume of modified Alsevier solution anticoagulant (Bachère et al 1988) and subsequently centrifuged for 10 min at 800×g, 4°C. Hemocyte pellets were immediately used for RNA extraction.…”

Section: Shrimp Sourcementioning

confidence: 99%

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Luan

Zhang

et al. 2009

Cell Stress and Chaperones

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Heat shock protein 90 (HSP90) works as a multifunctional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25°C to 35°C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

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Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation

Cited by 140 publications

References 20 publications

Lumhol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas

Lumhol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas

Evidence of a bactericidal permeability increasing protein in an invertebrate, theCrassostrea gigas Cg-BPI

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

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