Differences in membrane lipid composition and fluidity of transplanted GRSL lymphoma cells, depending on their site of growth in the mouse

GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane lipid fluidities of these cells and of normal lymphoid cells were estimated by fluorescence polarization, using the probe diphenylhexatriene and by measuring the (free) cholesterol/phospholipid molar ratio in whole cells. The results indicate that the membrane fluidity (reciprocal of the lipid structural order) of the lymphoma cells increases in the order of their location: peripheral blood less than spleen less than mesenterial lymph node less than ascites fluid. The membrane fluidities of normal lymphocytes from thymus, mesenterial lymph node and spleen were about the same, but higher than of peripheral blood lymphocytes, and between those of the lymphoma cells from lymph node and spleen. These results are confirmed by more extensive analysis on purified plasma membranes from the splenic and ascitic GRSL lymphoma cells and from normal splenocytes and thymocytes. The significantly higher lipid order parameter found in the GRSL plasma membrane isolated from the spleen as compared to those from the ascites cells could be fully explained by the differences measured in the major chemical determinants of the fluidity, i.e., the cholesterol/phospholipid ratio, the sphingomyelin content and the degree of saturation of the fatty acyl groups of the phospholipids. It was also found that the cholesterol/phospholipid ratio in erythrocyte membranes isolated from the peripheral blood of the tumor bearers was higher than in those from normal control mice. The observed differences in membrane fluidity between distinct subsets of tumor cells may be relevant to the sensitivity of these cells to immune attack or to drugs.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication

Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK(+)) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK(+) and, to a lesser extent, with wild-type c-Src, but not with kinase-dead c-Src. Mutation of residue Cx43 Tyr(265) (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK(+) in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43Delta263) that lacks residue Tyr(265). Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr(265), resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

Methodology going astray in population biology

This paper analyses the broad methodological structure of population-biological theorising. In it, I show that the distinction between initial exploratory, hypothesis-generating research and the subsequent process-reconstructing, hypothesis-testing type of research is not being made. Rather, the hypotheses generated in population biology are elaborated in such detail that students confound the initial research phase with the subsequent hypotheses-testing phase of research. In this context, I therefore analyse some testing procedures within the exploration phase and show that, as an extreme form of confusion, statistical null-models are mistakenly given the status of causal population-biological theory.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Cell division control by the Chromosomal Passenger Complex

The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.     

Framework for Modelling Economic Impacts of Invasive Species,Applied to Pine Wood Nematode in Europe

Background

Economic impact assessment of invasive species requires integration of information on pest entry, establishment and spread, valuation of assets at risk and market consequences at large spatial scales. Here we develop such a framework and demonstrate its application to the pinewood nematode, Bursaphelenchus xylophilus, which threatens the European forestry industry. The effect of spatial resolution on the assessment result is analysed.

Methodology/Principal Findings

Direct economic impacts resulting from wood loss are computed using partial budgeting at regional scale, while impacts on social welfare are computed by a partial equilibrium analysis of the round wood market at EU scale. Substantial impacts in terms of infested stock are expected in Portugal, Spain, Southern France, and North West Italy but not elsewhere in EU in the near future. The cumulative value of lost forestry stock over a period of 22 years (2008–2030), assuming no regulatory control measures, is estimated at €22 billion. The greatest yearly loss of stock is expected to occur in the period 2014–2019, with a peak of three billion euros in 2016, but stabilizing afterwards at 300–800 million euros/year. The reduction in social welfare follows the loss of stock with considerable delay because the yearly harvest from the forest is only 1.8%. The reduction in social welfare for the downstream round wood market is estimated at €218 million in 2030, whereby consumers incur a welfare loss of €357 million, while producers experience a €139 million increase, due to higher wood prices. The societal impact is expected to extend to well beyond the time horizon of the analysis, and long after the invasion has stopped.

Conclusions/Significance

Pinewood nematode has large economic consequences for the conifer forestry industry in the EU. A change in spatial resolution affected the calculated directed losses by 24%, but did not critically affect conclusions.