Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Effect of exercise duration on postprandial hypertriglyceridemia in men with metabolic syndrome.

We examined the effect of exercise on postprandial hypertriglyceridemia (PHTG) and insulin resistance in individuals with metabolic syndrome. Subjects were 10 hypertriglyceridemic men with insulin resistance [age = 35.0 +/- 1.8 yr, body weight = 90.7 +/- 3.3 kg, fasting triglyceride (TG) = 2.6 +/- 0.4 mmol/l, peak oxygen consumption ((.)Vo(2peak)) = 36.0 +/- 1.3 ml(-1).kg(-1).min(-1), and homeostatic model assessment of insulin resistance (HOMA-IR)= 3.1 +/- 0.3]. Each participant performed a control trial (Ctr; no exercise) and three exercise trials at 60% of their (.)Vo(2peak) for 30 min (30 min-Ex), 45 min (45 min-Ex) and 60 min (60 min-Ex). All subjects had a fat meal in each trial. In the exercise trials, the subject jogged on a treadmill for a designated duration of 12 h before ingestion of a fat meal. Blood samples were taken at 0 h (before the meal) and at 2, 4, 6, and 8 h after the meal. The plasma TG, area score under TG concentration curve over an 8-h period (TG AUC) after the meal, and HOMA-IR were analyzed. The TG AUC scores in both the 45 min-Ex and 60 min-Ex were 31 and 33% lower, respectively, than Ctr (P < 0.02). There were no significant differences in TG AUC scores between the 30 min-Ex and the Ctr (P > 0.05). There were no trial differences in the fasting plasma glucose concentration (P > 0.05). HOMA-IR values in the 30 min-Ex, 45 min-Ex, and 60 min-Ex trials were lower than the Ctr (P < 0.03), but no significant differences were found in HOMA-IR among the exercise trials. The results suggest that for physically inactive individuals with metabolic syndrome, exercising at moderate intensity for 45 min effectively attenuates PHTG while exercise for 30 min is sufficient to improve insulin action.     

Localization of Src homology 2 domain-containing phosphatase 1 (SHP-1) to lipid rafts in T lymphocytes: functional implications and a role for the SHP-1 carboxyl terminus

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Characterisation of atypical enteropathogenic E. coli strains of clinical origin

Background  

Enteropathogenic E. coli (EPEC) is a prominent cause of diarrhoea, and is characterised in part by its carriage of a pathogeniCity island: the locus for enterocyte effacement (LEE). EPEC is divided into two subtypes according to the presence of bundle-forming pili (BFP), a fimbrial adhesin that is a virulence determinant of typical EPEC (tEPEC), but is absent from atypical EPEC (aEPEC). Because aEPEC lack BFP, their virulence has been questioned, as they may represent LEE-positive Shiga toxin-producing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP. To determine if aEPEC isolated from humans in Australia or New Zealand fall into either of these categories, we undertook phylogenetic analysis of 75 aEPEC strains, and compared them with reference strains of EPEC and STEC. We also used PCR and DNA hybridisation to determine if aEPEC carry virulence determinants that could compensate for their lack of BFP.     

Coal Fly Ash Impairs Airway Antimicrobial Peptides and Increases Bacterial Growth

Air pollution is a risk factor for respiratory infections, and one of its main components is particulate matter (PM), which is comprised of a number of particles that contain iron, such as coal fly ash (CFA). Since free iron concentrations are extremely low in airway surface liquid (ASL), we hypothesize that CFA impairs antimicrobial peptides (AMP) function and can be a source of iron to bacteria. We tested this hypothesis in vivo by instilling mice with Pseudomonas aeruginosa (PA01) and CFA and determine the percentage of bacterial clearance. In addition, we tested bacterial clearance in cell culture by exposing primary human airway epithelial cells to PA01 and CFA and determining the AMP activity and bacterial growth in vitro. We report that CFA is a bioavailable source of iron for bacteria. We show that CFA interferes with bacterial clearance in vivo and in primary human airway epithelial cultures. Also, we demonstrate that CFA inhibits AMP activity in vitro, which we propose as a mechanism of our cell culture and in vivo results. Furthermore, PA01 uses CFA as an iron source with a direct correlation between CFA iron dissolution and bacterial growth. CFA concentrations used are very relevant to human daily exposures, thus posing a potential public health risk for susceptible subjects. Although CFA provides a source of bioavailable iron for bacteria, not all CFA particles have the same biological effects, and their propensity for iron dissolution is an important factor. CFA impairs lung innate immune mechanisms of bacterial clearance, specifically AMP activity. We expect that identifying the PM mechanisms of respiratory infections will translate into public health policies aimed at controlling, not only concentration of PM exposure, but physicochemical characteristics that will potentially cause respiratory infections in susceptible individuals and populations.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid