Field evaluation of the bait toxicant chlorfluazuron in eliminating Coptotermes acinaciformis (Froggatt) (Isoptera: Rhinotermitidae)

Two aspects of the Exterra Termite Interception and Baiting System (Ensystex, Fayetteville, NC) were evaluated in a field experiment using 13 termite mounds near Townsville, Australia. First, a cellulose-acetate powder containing either 0.05% wt:wt or 0.25% wt:wt chlorfluazuron (Requiem, Ensystex, Fayetteville, NC) was tested for its efficacy in eliminating colonies of the xylophagous mound-building subterranean termite Coptotermes acinaciformis (Froggatt). The moist bait matrix was replenished during the first inspection of 10 mounds (five mounds by two treatments) used in the experiment. Second, a single application of the moist bait matrix was used on three additional mounds to test termite responses and the effectiveness of 0.25% wt:wt chlorfluazuron. Although there was no evidence of repellence, there was little removal of replenished bait. Five colonies were eliminated by 0.05% wt:wt chlorfluazuron and five colonies by 0.25% wt:wt chlorfluazuron: another colony was moribund, and elimination appeared imminent. Colony decline was first suspected some 12 wk after bait application, and colony elimination was confirmed, by destructive sampling, about 5 wk later. Colony elimination may have occurred within 12 wk. One colony was an anomaly and did not succumb to the effects of the toxicant. Another colony was not eliminated because of invasion of the baiting system by ants. Ants, principally Iridomyrmex purpureus (F. Smith) group and Papyrius nitidus (Mayr) group, occurred commonly in the stations during the experiment. Microcerotermes sp. was found in five of the C. acinaciformis mounds, after colony elimination. Inspections of small sections of mounds and wooden dowels inserted into mounds were reliable methods for monitoring colony health.     

Hypertension in L-NAME-treated diabetic rats depends on an intact sympathetic nervous system

We demonstrated previously that induction of diabetes in rats that were treated chronically with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) causes a severe, progressive increase in mean arterial pressure. This study tested the role of the sympathetic nervous system in that response. Rats were instrumented with chronic artery and vein catheters and assigned randomly to four diabetic groups pretreated with vehicle (D), L-NAME (D+L), the alpha(1)- and beta-adrenergic receptor antagonists terazosin and propranolol (D+B), or L-NAME, terazosin, and propranolol (D+LB). After baseline measurements were taken, rats were pretreated; 6 days later, streptozotocin was administered and 3 wk of diabetes ensued. D+L rats had a marked, progressive increase in arterial pressure that by day 20 was approximately 60 mmHg greater than in D rats. The pressor response to L-NAME was significantly attenuated in diabetic rats cotreated with adrenergic blockers. During week 1 of diabetes, plasma renin activity (PRA) increased and then returned to control levels in D rats. PRA increased progressively in D+L rats, and chronic adrenergic receptor blockade restored the biphasic renin response in D+LB rats. These results suggest that the sympathetic nervous system may be involved in the hypertensive response to onset of diabetes in L-NAME-treated rats, possibly through control of renin secretion.     

Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132-140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs (n = 7), acute ductal constriction initially increased PBF by 627% (P < 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity (P < 0.05) and a 106% increase in plasma ET-1 levels (P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ET(B) receptor protein levels. The infusion of PD-156707 (an ET(A) receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ET(A) receptor-mediated vasoconstriction and an ET(A) receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.     

Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I

The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.     

Identification of the phosphotyrosine proteome from thrombin activated platelets

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.     

Identification of a phosphofructokinase-encoding gene from Streptococcus thermophilus CNRZ1205--a novel link between carbon metabolism and gene regulation?

In order to isolate genes encoding so-called Two-Component Regulatory Systems from the lactic acid bacterium Streptococcus thermophilus, a cloning strategy was employed based on suppression of the alkaline phosphatase-negative phenotype displayed by the Escherichia coli strain ANCC22. Several suppressing clones were obtained which were shown to produce alkaline phosphatase activity. Sequence analysis of four of these clones revealed the presence of overlapping DNA inserts representing two ORFs, designated pfkT and pykT, whose deduced protein products exhibit significant similarity to phosphofructokinases and pyruvate kinases, respectively, from a variety of bacteria. A plasmid bearing pfkT was shown to complement a phosphofructokinase-negative mutant of E. coli, showing that this gene indeed specifies phosphofructokinase activity. It was shown that suppression of the alkaline phosphatase-negative phenotype of E. coli ANCC22 due to the presence of pfkT is caused by modulation of the intracellular level of acetyl phosphate.     

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters

AIMS: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. METHODS AND RESULTS: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. CONCLUSIONS: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. SIGNIFICANCE AND IMAPCT OF THE STUDY: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.     

Accidental altruism in insular pit-vipers (Gloydius shedaoensis, Viperidae)

Darwinian theory predicts that organisms will display traits that benefit themselves rather than other individuals; exceptions to this rule usually are explicable by kin selection. Our studies on an insular population of venomous snakes in north-eastern China reveal a different situation. Only one species of snake (Gloydius shedaoensis, Viperidae) occurs on the island of Shedao, and displays altruism between size (age) classes. First, small snakes frequently kill prey items larger than they can swallow themselves. This behaviour enhances rates of feeding of larger conspecifics, which scavenge the birds' carcasses. Second, large snakes kill raptorial birds (sparrowhawks Accipiter nisus) that pose little or no threat to themselves. This behaviour reduces predation risk for smaller snakes. These effects are presumably accidental consequences of the high venom toxicity of the pit-vipers, which enable them to kill inedible prey and non-threatening predators at little cost. Nonetheless, this accidental altruism may have significant ecological consequences. For example, these behaviours may contribute to the remarkably high population densities of snakes on Shedao.     

Investigation of protein export in Bifidobacterium breve UCC2003

The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.