Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17 degrees C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae

The N-acyl homoserine lactone (AHL)-mediated quorum-sensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P syringae were examined. Both an aefR- mutant and an ahlI- ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in E syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.     

Parathyroid hormone-related protein production in the lamprey <Emphasis Type="Italic">Geotria australis</Emphasis>: developmental and evolutionary perspectives

This study explored the distribution of parathyroid hormone-related protein (PTHrP) and its mRNA in tissues of the lamprey Geotria australis, a representative of one of the two surviving groups of an early and jawless stage in vertebrate evolution. For this purpose, antibodies to N-terminal and mid-molecule human PTHrP were used to determine the locations of the antigen. Sites of mRNA production were demonstrated by in situ hybridisation with a digoxigenin-labelled riboprobe to exon VI of the human PTHrP gene. The results revealed that antigen and its mRNA were widely distributed among similar sites of tissue localisation to those described for mammalian and avian species. However, some novel sites of localisation, such as in the gill and notochord, were also found. Some differences in PTHrP localisation were noted among individuals at different intervals of the life cycle, indicating that the distributions of PTHrP, and possibly its roles, change with the stage of development in this species. The widespread tissue distribution in G. australis implies diverse physiological roles for this protein. The presence of PTHrP in the lamprey, a representative of a group of vertebrates, which apparently evolved over 540 million years ago, strongly suggests that it is a protein of ancient origin. In addition, the successful use of antibodies and probes based on the human sequence in the lamprey also provides evidence that the PTHrP molecule may have been conserved from lampreys through to humans.     

Regulation of lung injury and repair by Toll-like receptors and hyaluronan

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.     

An approach to heterobifunctional poly(ethyleneglycol) bioconjugates

A family of differentially substituted poly(ethyleneglycol) building blocks has been assembled from commercially available material. Their utility is demonstrated by formation of amino acid conjugates, image contrast agents, gold nanoparticles, and functional antibody conjugates. Application in the cellular trafficking of antitumoral agent conjugates is expected.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette-Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell-mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium-specific IFN-gamma-secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid-encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyer's patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium-specific IFN-gamma production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid-encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.     

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.     

N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis

Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.     

Purification of vitellin from the ovary of Chinese mitten-handed crab (Eriocheir sinensis) and development of an antivitellin ELISA

Vitellin was purified from ovaries of mature female Chinese mitten-handed crab (Eriocheir sinensis) using gel filtration chromatography. Analysis by native PAGE showed the vitellin had a native molecular mass of 520 kDa, while denaturing SDS-PAGE revealed two subunits of 97 and 74 kDa. Purified vitellin was used to raise polyclonal antisera, with which an enzyme-linked immunosorbent assay (ELISA) was developed. The ELISA was sensitive and could effectively detect vitellin in the range of 7.8-500 ng. Furthermore, vitellin levels in various developmental stages of oogenesis were measured with the ELISA assay. The results indicated that levels of vitellin increased significantly from 0.22 mg/ovary at Stage II to 360.31 mg/ovary at Stage IV.