Development and characterisation of a large diameter decellularised vascular allograft

The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg^?1 total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft.     

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered expression of fructosyltransferases

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+?(gene of interest positive) and GOI? (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+?and GOI? for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI? compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI?. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.     

Feeling the pressure at home: Predator activity at the burrow entrance of an endangered arid‐zone skink

Habitat modification and invasive species are among the most important contemporary drivers of biodiversity loss. These two threatening processes are often studied independently and few studies have focused on how they interact to influence species declines. Here we assess the predation pressure placed on the threatened great desert skink (Liopholis kintorei) and how this interacts with fire‐induced habitat modifications. We collected daily track data of potential predators for 1 month at 30 great desert skink burrow‐systems where vegetation cover varied significantly after experimental burns. We used these data to evaluate potential predation pressure at the burrow‐system and assess whether fire influenced predator pressure. We supplemented this analysis by documenting predation via the inspection of large mammalian predator scats collected from great desert skink habitat. The level of feral cat activity at a burrow‐system entrance was significantly higher than that of any other potential predator, however fire had no effect on the visitation rates of feral cats, dingoes or large snakes to great desert skink burrow‐systems. The remains of great desert skink were found significantly more frequently in feral cat scats, compared to fox and dingo scats. We provide the first direct evidence that feral cats are a significant predator for great desert skink, thus supporting the hypothesis that feral cat predation is a key threatening process. Feral cat activity was not influenced by small‐scale experimental burns, however, this does not preclude an effect of larger scale fires and we recommend further research exploring this possible interaction.     

Effects of male paragonial gland secretion on sexual receptivity and courtship behaviour of female Drosophila melanogaster

Female Drosophila melanogaster show a switch-off in sexual receptivity and an increase in oviposition rate following impregnation. These effects are also observed in unfertilized virgin females which receive transplants of intact male paragonia into the abdominal body cavity. Virgin Amherst wildtype females show a repertoire of responses to male courtship characterized by high rates of kicking and fending and a low rate of genital extrusion. Fertilized females show low rates of kicking and fending but a high rate of genital extrusion. Virgin female hosts receiving paragonial gland implants show an altered pattern of courtship rejection with low rates of kicking and fending. Studies on these host females, and on females homozygous for the mutant allele female-sterile, suggest that the expression of genital extrusion depends, in addition to the paragonial gland substance, upon additional information input from abdominal stretch receptors or from the ovary.     

Tightly bound oxalacetate and the activation of succinate dehydrogenase

Soluble succinate dehydrogenase prepared from acetone powders of submitochondrial particles is almost entirely in the deactivated state and contains 0.5 mole of oxalacetate (OAA) per mole of histidyl flavin. OAA is dissociated by succinate, malonate, IDP, ITP, and high concentrations of anions at elevated temperatures, but not significantly in the cold, with concurrent activation of the enzyme; the high energy of activation observed for OAA release and for activation suggests that a conformation change in the protein is involved. On removal of OAA, a reversible activation-deactivation cycle dependent on the pH is demonstrable. Submitochondrial particles behave similarly but appear to contain 1 mole of tightly bound OAA per histidyl flavin in the deactivated state.     

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.     

Monoclonal anti-Id antibodies react with varying proportions of human B lineage cells

Monoclonal antibodies to idiotypic determinants are being used with increasing frequency for analysis and treatment of B cell malignancies. In the present study we have compared the idiotypic specificities of a panel of 39 mouse monoclonal anti-idiotype (anti-Id) antibodies developed against 16 monoclonal human immunoglobulins (Ig). The Id cross-reactivities of these antibodies with Ig products of normal and abnormal B cells were examined by immunofluorescence and immunochemical methods. The reactivity patterns of these anti-Id antibodies with a normal population of plasma cells were highly variable in the immunofluorescence assay. Six were reactive with 2 to 10% of normal plasma cells, 30 with 0.1 to 2% of plasma cells, and three with less than 0.1% of plasma cells from blood, bone marrow, spleen, or tonsils. These reactivity patterns were relatively consistent among samples from 23 Caucasian, black, and Oriental adults. Although the reactivities of most anti-Id antibodies in the panel were not restricted to a particular Ig isotype, several were preferentially reactive with a particular heavy or light chain isotype: one IgM-, two IgA-, two kappa-, and three lambda-restricted antibodies. The immunofluorescence data was confirmed by biosynthetic analysis of Id+ molecules produced by a normal plasma cell population. When the reactivity of this panel of anti-Id antibodies with nonhomologous B cell neoplasms was examined, seven of 30 myelomas or leukemia-derived products and one of nine B cell leukemias or lymphomas without paraproteins were found to be cross-reactive with one or two of the anti-Id antibodies. Although clearly significant, the cross-reactivity between the Id of these paraproteins appeared to be of lower affinity than the reactivity of the homologous Id with their respective anti-Id antibodies. The results reveal a remarkable diversity in the specificities of monoclonal antibodies classified by conventional criteria as anti-Id antibodies, and indicate the potential usefulness of a panel of antibodies for analyzing clonal diversity in normal and abnormal B cell development.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.