Calcium buffer solutions and how to make them: a do it yourself guide.

In measurements of the intracellular free calcium concentration ([Ca2+]) using either microelectrodes or fluorescent probes, calibration is normally carried out in EGTA calcium buffer solutions. In the first part of the article the general properties of calcium buffer solutions are discussed, the equations used to calculate the apparent calcium binding constant (Kapp) are derived, and the difficulties in the calculation are discussed. The effects of the purity of EGTA as well as the influence of calcium contamination on the buffer solutions are explained. Because of the difficulties in calculating Kapp, and the importance of EGTA purity and calcium contamination, it is suggested that it is easier to measure all three under the appropriate experimental conditions using the method of Bers (1982). In the second part a do-it-yourself guide to the preparation of EGTA calcium buffer solutions is given. An experimental example is provided using the Bers method to measure purity, contamination, and Kapp. It is concluded that unless all three factors are known it is not possible to prepare accurate EGTA calcium buffer solutions.     

Insights into autotaxin: how to produce and present a lipid mediator

Autotaxin (ATX) is a secreted phosphodiesterase that produces the lipid mediator lysophosphatidic acid (LPA). LPA acts through specific guanine-nucleotide-binding protein (G protein)-coupled receptors to stimulate migration, proliferation, survival and other functions in many cell types. ATX is important in vivo for processes as diverse as vasculogenesis, lymphocyte trafficking and tumour progression. However, the inner workings of ATX have long been elusive, in terms of both its substrate specificity and how localized LPA signalling is achieved. Structural studies have shown how ATX recognizes its substrates and may interact with the cell surface to promote specificity in LPA signalling.     

Autophagy and T-cell education in the thymus: Eat yourself to know yourself

During intrathymic generation of the T cell repertoire, a series of selection processes ensure that only self-MHC (Major Histocompatibility Complex) restricted and self-tolerant T cells are allowed to survive. Interactions with MHC ligands on the surface of thymic epithelial cells (TECs) play a pivotal role in the decision-making of developing thymocytes. A number of distinct cell-biological features of TECs have emerged that may predispose them to serve non-redundant functions in thymocyte “education”. Thus, cortical TECs express a rather unique set of proteolytic enzymes for antigen processing in the context of positive selection, whereas medullary TECs "ectopically" express a plethora of otherwise strictly tissue-restricted antigens (TRAs), a property that obviously has evolved to make these self-antigens "visible" to developing thymocytes for negative selection. One of the latest additions to this growing list of functional adaptations of TECs is their constitutively high rate of autophagy. Recently, we have provided evidence that autophagy in TECs shuttles cytoplasmic self-antigens into the MHC class II loading pathway for positive selection of T cells and tolerance induction.     

Quantifying elasticity analysis: how external effectors cause changes to metabolic systems

The sites of action of external effectors, such as inhibitors or hormones, on metabolic systems can be described qualitatively by elasticity analysis, or quantitatively by regulation analysis. The use of the latter approach has been limited, due to its practical complexity. In this study, we report mathematical relationships that relate the finite changes in system variables (fluxes and metabolite concentrations) to changes in activity of metabolic processes brought about by a single step addition of an effector. The activation or inhibition of a process by an effector is measured from changes in flux and intermediate levels. The changes in activity of each process can be used to describe, semi-quantitatively, which activations or inhibitions of the system processes are important in bringing about the observed levels of system variables.     

Relationships betweenApanteles flavipes (Hym.: Braconidae) andNosema bordati (Microspora,Nosematidae) parasites ofChilo partellus (Lep.: Pyralidae)

A laboratory study was conducted on the relationships betweenNosema bordati Goudegnon andApanteles flavipes (Cameron) when they simultaneously parasitiseChilo partellus (Swinhoe). The results indicate thatN. bordati is highly pathogenic to the immature instars of the parasitoid. Lightly infected adults ofA. flavipes are able to transmit the Microsporidian.     

Mammalakis n. g. and Mammalakinae n. subfam. (Nematoda: Heterakoidea: Kiwinematidae): parasites of mole rats (Rodentia: Bathyergidae and spalacidae)

Heterakis macrospiculum Ortlepp, 1939 is redescribed as the type species of Mammalakis n. g., known only from mole rats, and characterised by an absence of cheilostomal flanges to the lips, and the presence of caudal expansions of the lateral alae on males and of massive spicules. H. spalacis Marcu, 1930 and H. spalaxi Kozlov & Jangolenko, 1962 are also referred to the new genus for which the Mammalakinae n. subfam. of the Kiwinematidae is erected. The new evidence, while not clarifying phylogenetic relationships within the Heterakoidea, supports the view that that superfamily had its origins before the separation of Laurasia and Gondwanaland.     

Solving the problem of how to eat something as big as yourself: Diverse bacterial strategies for degrading polysaccharides

Polysaccharide digestion by bacteria is an important activity in many ecosystems, and a number of bacterial genera can perform this function. Although many papers have been published about the properties of isolated polysaccharide-degrading enzymes, relatively little is known about how intact bacteria degrade polysaccharides. This review summarizes recent findings suggesting that there are at least three different strategies. the most familiar one is the excretion of extracellular polysaccharidases, which diffuse to and degrade nearby polysaccharides. An example of this type of strategy is provided by the plant pathogen,Erwinia spp. A second strategy is to have the enzyme exposed to the extracellular medium but attached to the surface of the cell. Examples of this strategy are provided by the pullulanase system ofKlebsiella oxytoca and the cellulosomes ofClostridium thermocellum. A strategy that could be seen as a combination of the extracellular enzyme strategy and the surface organelle strategy is provided byVibrio harveyi, which attaches to its substrate, chitin, via proteins that appear to be specialized for attachment and produces extracellular enzymes that attack the chitin. A third strategy is to import the polysaccharide, as appears to be done byBacteroides spp. In this instance, the polysaccharide is bound to an outer membrane receptor, then passes into the periplasm where the degradative enzymes are located. The ecological advantages and disadvantages of these systems are discussed, and areas where further research is needed are defined.     

Survey for parasites ofSesamia calamistis (Lep.: Noctuidae) andEldana saccharina (Lep.: Pyralidae) in southwestern Nigeria

Field surveys were conducted during 1990–92 to document the relative abundance of different species of parasites of the lepidopterous stem borersSesamia calamistis Hampson andEldana saccharina Walker in maize fields in southwestern Nigeria. Species of parasitoids detected on both stem borers included the larvalpupal parasitoidsSturmiopsis parasitica Curran (Diptera: Tachinidae) andBrachymeria feae Masi (Hymenoptera: Chalcididae), and the larval parasitoidDolichogenidea polaszeki Walker (Hymenoptera: Braconidae). The braconidCotesia sesamiae (Cameron) was found attackingS. calamistis. The hyperparasitoidExoristobia dipterae (Risbec) (Hymenoptera: Encyrtidae) was detected on a pupa ofS. parasitica. Parasitic nematodes belonging toMermis sp. and/orHexamermis sp. were found infesting larvae of both stem borers. Overall, larval/pupal parasitization levels at Ibadan were low and ranged from 4.2 to 22.8% forS. calamistis and 1.2 to 13% forE. saccharina. Of the parasites found,S. parasitica was the most common, followed by nematodes. Four hymenopteran egg parasitoids were found attackingS. calamistis: Telenomus busseolae Gahan,T. isis Polaszek (Scelionidae),Lathromeris ovicida Risbec, andTrichogrammatoidea eldanae Viggiani (Trichogrammatidae). Egg parasitization ranged from 13.4 to 41.5%. The only egg parasitoid detected onE. saccharina wasTelenomus applanatus Bin and Johnson, which inflicted only 5% parasitization.     

Prebiotic replicase evolution in a surface-bound metabolic system: parasites as a source of adaptive evolution

Background  

The remarkable potential of recent forms of life for reliably passing on genetic information through many generations now depends on the coordinated action of thousands of specialized biochemical "machines" (enzymes) that were obviously absent in prebiotic times. Thus the question how a complicated system like the living cell could have assembled on Earth seems puzzling. In seeking for a scientific explanation one has to search for step-by-step evolutionary changes from prebiotic chemistry to the emergence of the first proto-cell.