ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

Energy-dispersive X-ray fluorescence spectrometry as a tool for zinc, iron and selenium analysis in whole grain wheat

Background and aims

Crop biofortification programs require fast, accurate and inexpensive methods of identifying nutrient dense genotypes. This study investigated energy-dispersive X-ray fluorescence spectrometry (EDXRF) for the measurement of zinc (Zn), iron (Fe) and selenium (Se) concentrations in whole grain wheat.

Methods

Grain samples were obtained from existing biofortification programs. Reference Zn, Fe and Se concentrations were obtained using inductively coupled plasma optical emission spectrometry (ICP-OES) and/or inductively coupled plasma mass spectrometry (ICP-MS). One set of 25 samples was used to calibrate for Zn (19–60?mg?kg^–1) and Fe (26–41?mg?kg^–1), with 25 further samples used to calibrate for Se (2–31?mg?kg^–1 ). Calibrations were validated using an additional 40–50 wheat samples.

Results

EDXRF limits of quantification (LOQ) were estimated as 7, 3 and 2?mg?kg^–1 for Zn, Fe, and Se, respectively. EDXRF results were highly correlated with ICP-OES or -MS values. Standard errors of EDXRF predictions were ±2.2?mg Zn kg^–1, ±2.6?mg Fe kg^–1, and ±1.5?mg Se kg^–1.

Conclusion

EDXRF offers a fast and economical method for the assessment of Zn, Fe and Se concentration in wheat biofortification programs.     

Experimental beetle metapopulations respond positively to dynamic landscapes and reduced connectivity

Interactive effects of multiple environmental factors on metapopulation dynamics have received scant attention. We designed a laboratory study to test hypotheses regarding interactive effects of factors affecting the metapopulation dynamics of red flour beetle, Tribolium castaneum. Within a four-patch landscape we modified resource level (constant and diminishing), patch connectivity (high and low) and patch configuration (static and dynamic) to conduct a 2(3) factorial experiment, consisting of 8 metapopulations, each with 3 replicates. For comparison, two control populations consisting of isolated and static subpopulations were provided with resources at constant or diminishing levels. Longitudinal data from 22 tri-weekly counts of beetle abundance were analyzed using bayesian Poisson generalized linear mixed models to estimate additive and interactive effects of factors affecting abundance. Constant resource levels, low connectivity and dynamic patches yielded greater levels of adult beetle abundance. For a given resource level, frequency of colonization exceeded extinction in landscapes with dynamic patches when connectivity was low, thereby promoting greater patch occupancy. Negative density dependence of pupae on adults occurred and was stronger in landscapes with low connectivity and constant resources; these metapopulations also demonstrated greatest stability. Metapopulations in control landscapes went extinct quickly, denoting lower persistence than comparable landscapes with low connectivity. When landscape carrying capacity was constant, habitat destruction coupled with low connectivity created asynchronous local dynamics and refugia within which cannibalism of pupae was reduced. Increasing connectivity may be counter-productive and habitat destruction/recreation may be beneficial to species in some contexts.