Monoclonal antibodies which identify a genus-specific Listeria antigen.

Fifteen murine monoclonal antibodies (MAbs) which react specifically with a protein antigen found in all species of Listeria were developed and characterized. These MAbs were tested extensively by both enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses for cross-reaction with non-Listeria organisms, such as Staphylococcus, Streptococcus, Citrobacter, Pseudomonas, and Salmonella species, and were found to be nonreactive. The genus-specific antigen was identified as a heat-stable protein with a molecular weight in the range of 30,000 to 38,000 (under both reducing and nonreducing conditions), depending on the species of Listeria tested. In Listeria monocytogenes, L. innocua, L. ivanovii, and L. seeligeri the antigen has a molecular weight of approximately 30,000 to 34,000. In L. grayi and L. murrayi it has a molecular weight of approximately 35,000 to 38,000. In addition, several of the MAbs recognize lower-molecular-weight protein bands. There appear to be at least two groups of Listeria-specific MAbs based upon isotype and results of enzyme-linked immunosorbent assay and Western blot analyses. These MAbs have proven to be useful in the development of a diagnostic assay for Listeria species in food products.     

A study of cell electrophoresis as a means of purifying growth hormone secreting cells

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partially purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flows electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.     

Of mast and mean: differential‐temperature cue makes mast seeding insensitive to climate change

Mast‐seeding plants often produce high seed crops the year after a warm spring or summer, but the warm‐temperature model has inconsistent predictive ability. Here, we show for 26 long‐term data sets from five plant families that the temperature difference between the two previous summers (ΔT) better predicts seed crops. This discovery explains how masting species tailor their flowering patterns to sites across altitudinal temperature gradients; predicts that masting will be unaffected by increasing mean temperatures under climate change; improves prediction of impacts on seed consumers; demonstrates that strongly masting species are hypersensitive to climate; explains the rarity of consecutive high‐seed years without invoking resource constraints; and generates hypotheses about physiological mechanisms in plants and insect seed predators. For plants, ΔT has many attributes of an ideal cue. This temperature‐difference model clarifies our understanding of mast seeding under environmental change, and could also be applied to other cues, such as rainfall.     

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

Calcium dynamics in the failing heart: restoration by beta-adrenergic receptor blockade

Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using beta-adrenergic receptor antagonists (beta-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of beta-blocker treatment on Ca2+ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective beta-receptor blocker (5 mg. kg-1. day-1 propranolol) for 2 wk. Ca2+ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca2+ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca2+ kinetic measurements of time to peak and time decay of the caffeine-induced Ca2+ transient to 50% relaxation were also normalized. Immunoblot analysis of untreated TOT heart samples showed a 3.6-fold reduction of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), whereas Na+/Ca2+ exchanger (NCX) concentrations were increased 2.6-fold relative to nontransgenic samples. Propranolol treatment of TOT mice reversed the alterations in SERCA and NCX protein levels but not potassium channels. Although restoration of Ca2+ dynamics occurred within 2 wk of beta-blockade treatment, evidence of functional improvement in cardiac contractility assessed by echocardiography took 10 wk to materialize. These results demonstrate that beta-adrenergic blockade restores Ca2+ dynamics and normalizes expression of Ca2+-handling proteins, eventually leading to improved hemodynamic function in cardiomyopathic hearts.     

Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.     

中国的炭疽杆菌DNA分型及其地理分布

炭疽广泛分布于中国各地,特别是西部地区,并经常造成人畜疾病,在一项合作研究中,用多位点VNTR分析（MLVA）对从1952-1998年自中国主要地理流行区域分离的病人,病畜和土壤等来源的炭疽杆菌进行了基因分型,MLVA分析结果揭示了21种新的基因型,其等位基因组合在以前世界范围分离物的研究中未曾发现,此外,分离物的分群显示,A3b组是地理上最广泛分布的基因组,说明该组可能是中国的“地方流行株”。而来自古丝绸之路重要贸易中心新疆的大量分离株其基因型特别分散。     

The role of endothelial calcium and nitric oxide in the localisation of atherosclerosis

A mathematical model of endothelial cell calcium signalling and nitric oxide synthesis under flow conditions is presented. The model is coupled to two important environmental stimuli for endothelial cells: the frictional shear stress exerted on the cell membrane by the blood flow; and the binding of adenosine triphosphate in the bloodstream to cell surface receptors. These stimuli are closely linked to haemodynamic flow conditions and are, in general, spatially varying, allowing the cellular response in different regions of the endothelium to be evaluated. This is used to indicate which areas of the artery wall experience reduced bioavailability of nitric oxide, which is a major factor in the onset of atherosclerosis. The model thus directly addresses the key issue of the causative link, and its underlying biochemical mechanisms, between incidence of atherosclerosis and regions of low wall shear stress (WSS). Model results show that intracellular levels of free calcium and endothelial nitric oxide synthase are lower in endothelial cells adjacent to a region of recirculating flow than in cells adjacent to regions of fully developed arterial flow. This will lead to deficient levels of nitric oxide in the recirculation zone and hence a potentially elevated risk of developing atherosclerotic plaque. This is consistent with the observed spatial correlation between atherosclerosis and regions of disturbed blood flow and low WSS, and provides a mechanism for the localisation of the disease to sites such as arterial bifurcations and bends.     

The effect of competition on species' distributions depends on coexistence,rather than scale alone

One of the key problems in ecology is our need to anticipate the set of locations in which a species will be found (hereafter species' distributions). A major source of uncertainty in these models is the role of interactions among species (hereafter biotic interactions). Unfortunately, it is difficult to directly study this problem at large spatial scales and we lack a clear understanding of when biotic interactions shape species' distributions. We show a simple, direct link between the ease of species' coexistence and the importance of competition for shaping species' distributions. We show that increasing the ease of species' coexistence reduces the influence of biotic interactions. Changing the spatial scale of the analysis can reduce the influence of species interactions, but only when it promotes regional coexistence. Using these ideas, we analyze the conditions under which biotic interactions alter species' distributions in a Lotka–Volterra model of competition along an environmental gradient and argue that coexistence theory, rather than scale alone, provides a guide to the influence of species interactions. Our results provide a guide to the facets of biotic interactions that are necessary to anticipate their effects on species distributions. As such, we expect our work will help the development of more realistic distribution models.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.