Towards a simple logic in the determination of biological groups: the species and supraspecific groups

In this paper we discuss about the utility of the species concept as real definition, particularly the Mayr concept. We propose a method for the logical separation of taxa based in the statements of the logical mathematics and the application of the sets theory to the concepts in systematic. We attempt to provide an objective methodology for the interpretation of natural groups in biology including the species as a basic group in evolution. We introduce the concept of the hypothetical ancestor as a mathematical possibility derived from the use of matrix calculations for non square matrix.     

Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics

Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.     

ARF6-dependent interaction of the TWIK1 K+ channel with EFA6, a GDP/GTP exchange factor for ARF6

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.     

Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.     

Effect of support materials on antibiotic MSW2000 production by immobilized Streptomyces violatus

The production of an antibiotic by free and immobilized cells of Streptomyces violatus through entrapment or adsorption on different materials was investigated. S. violatus entrapped in Ca-alginate beads gave low antibiotic activity compared to the free cell or adsorbed cell, while the adsorption of S. violatus on sponge cubes yielded the highest antibiotic concentration after 4 days of incubation in static cultures. A starch concentration of 10 g/L was optimum for the production of the antibiotic by adsorbed cells. The weight and size of the sponge cubes used for immobilization influenced production of the antibiotic and the optimum weight and size of the sponge were 0.8 g and 1.0 cm(3), respectively, yielding a maximum antibiotic production of 280 mg/ml. Maximum antibiotic production was obtained at an initial pH value of 7.5 and in an inoculum size of 3 ml (spore suspension) per 50 ml. The production of the antibiotic in a fixed-bed bioreactor reached a maximum value after 2 days of incubation at a circulation rate of 30 ml/h. The immobilized cells in the bioreactor were reused seven successive times over a period of 14 days.     

Effect of resuscitative mild hypothermia on glutamate and dopamine release,apoptosis and ischaemic brain damage in the endothelin-1 rat model for focal cerebral ischaemia

The relationship between glutamate and dopamine release, apoptosis and ischaemic damage was studied following induction of transient focal cerebral ischaemia under normothermic (37 degrees C) and postischaemic (resuscitative) mild hypothermic (34 degrees C for 2 h) conditions in sevoflurane anaesthetized male Wistar rats. Focal ischaemia was induced by infusing endothelin-1 adjacent to the middle cerebral artery. In vivo microdialysis was used to sample glutamate and dopamine from striatum and parietal cortex of the ipsilateral hemisphere. The volume of ischaemic damage and the degree of apoptosis were determined 24 h after the insult. In both striatum and cortex of the normothermic group an initial increase in extracellular glutamate and dopamine levels following endothelin-1 infusion was observed. Striatal glutamate levels remained enhanced (250% of baseline) throughout the experiment, while the other neurotransmitter levels returned to baseline values. Hypothermia significantly attenuated the endothelin-1 induced glutamate release in the striatum. It also reduced apoptosis and infarct volume in the cortex. These results indicate that: (i) postischaemic mild hypothermia exerts its neuroprotective effect by inhibiting apoptosis in the ischaemic penumbral region; and (ii) this effect is not associated with an attenuation of glutamate or dopamine release in the cortex.     

Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH(2)-terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

From one well to 9000: using high-density streptavidin-coated membranes for kinase detection

The study of kinases and their role in cellular regulation continues to expand as the human genome is sequenced and new kinases are identified as expression products of newly discovered genes. Reagents and assay systems that allow for sensitive, accurate, and high-throughput analysis of both purified kinases as well as crude extracts will enhance the characterization of these important cellular components and will speed the identification of appropriate therapeutic targets and the development of new and more effective treatments.     

The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.