Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM. At higher glcout another step, either transport over the glycosomal membrane or phosphorylation by hexokinase became rate-limiting. Mathematical modeling assuming that glucose uptake occurs by facilitated diffusion followed by an enzymatic step accurately predicts the experimental data. As predicted by the model, the internal concentration of non-metabolized glucose remains low till glcout = 5 mM and increases at higher external concentrations. In contrast to glucose, glycerol entered the cell by simple diffusion. Externally supplied glycerol did not affect glucose metabolism but externally added glucose interfered with glycerol metabolism in a way that suggests that the rate-limiting step is at the level of glycerol kinase. Our observations suggest that the bloodstream form of T. brucei adapts its glucose transport in a way that gives maximum yield at minimum expense.     

Habitat use of arctic charrSalvelinus alpinus in Thingvallavatn,Iceland

Synopsis Habitat use by four morphs of arctic charr,Salvelinus alpinus, was investigated in Thingvallavatn, Iceland, by sampling with pelagic and benthic gill nets. Sampling was done in May/June and August/September. Greatest abundance of fish was recorded in the littoral and epipelagic zone in early autumn. Catches were low in early summer. The four morphs are partly segregated in habitat. Small (SB-) and large benthivorous (LB-) charr have a more restricted spatial distribution than piscivorous (PI-), and especially planktivorous (PL-) charr. Both benthivorous morphs are mainly found in the littoral zone, and occur in largest numbers in stony shallows at depths between 0 and 10 m. PL-charr usually dominates in numbers in all habitats. PI-charr is most abundant in epibenthic habitats, although numbers are always low. All morphs are caught in higher numbers at night than during the day, but the diurnal activity difference is highest among SB-charr. The habitat use by different morphs is as may be expected from their morphology and diets. Within the population of PL-charr, young and small fish are more abundant on the bottom than in the pelagic zone, and there is a surplus of females in the pelagic zone. Along the benthic profile, young, small and immature PL-charr are more abundant in deep than in shallow waters. The results are discussed in relation to food supply, competition and predation. Possible reasons for the occurrence of four arctic charr morphs are also discussed.Contribution from the Thingvallavatn project.     

Stable Transformation of Embryogenic Tissues of <Emphasis Type="Italic">Pinus nigra</Emphasis> Arn. using a Biolistic Method

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l⁻¹. Resistant embryogenic tissue recovery required 10–12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.     

Copper(II) complexes of some N-substituted bis(aminomethyl)phosphinate ligands. An integrated EPR study of microspeciation and coordination modes by the two-dimensional simulation method

Copper(II) complexes of bis(aminomethyl)phosphinic acid (L1), bis(N-glycino-N-methyl)phosphinic acid (L2), bis(N-benzylglycino-N-methyl)phosphinic acid (L3), bis(l-prolino-N-methyl)phosphinic acid (L4) and bis(iminodicarboxymethyl-N-methyl)phosphinic acid (L5) were studied in aqueous solution by pH-potentiometric and electron paramagnetic resonance (EPR) spectroscopic methods. The EPR spectrum packages recorded at various ligand-to-metal concentration ratios and pH's were analyzed (after matrix rank analysis by the method of residual intensities as a complementary method) by the two-dimensional computer simulation method, which simultaneously determines the formation constants and the EPR parameters of the various (micro)species. L1 forms mono and bis complexes in different protonation states; for the other ligands, the mono complexes are always prevalent. For steric reasons, the formation of CuL is shifted to increasingly higher pH regions in the sequence L2, L3 and L4. CuLH was identified for L3, L4 and L5, and also CuLH(2) for L4 and L5. Cu(2)L(2) was found in small amounts for L3 and L4, while it predominates at pH>4 for L5. For L5, Cu(2)L(2)H(2) was also detected. For the ligands that form dimeric metal complexes in equimolar solution or at a ligand excess, Cu(2)L is formed at a metal ion excess. Ligation of the phosphinate O was suggested by indirect proofs in the protonated complexes of L1. For the ligands L2, L3 and L4, the copper(II) coordination in various species in different protonation states is reminiscent of that in the mono and bis complexes of simple amino acids. For the bis(aminomethyl)phosphinates, however, the cis positions of the amino groups in CuL are ensured by the structure of the ligand, and the isomers differ from each other in the (equatorial or axial) position of the second carboxylate group.     

From individual leaf elongation to whole shoot leaf area expansion: a comparison of three Aegilops and two Triticum species

BACKGROUND AND AIMS: Rapid leaf area expansion is a desirable trait in the early growth stages of cereal crops grown in low-rainfall areas. In this study, the traits associated with inherent variation in early leaf area expansion rates have been investigated in two wheat species (Triticum aestivum and T. durum) and three of its wild relatives (Aegilops umbellulata, A. caudata and A. tauschii) to find out whether the Aegilops species have a faster leaf area expansion in their early developmental stage than some of the current wheat species. METHODS: Growth of individual leaves, biomass allocation, and gas exchange were measured on hydroponically grown plants for 4 weeks. KEY RESULTS: Leaf elongation rate (LER) was strongly and positively correlated with leaf width but not with leaf elongation duration (LED). The species with more rapidly elongating leaves showed a faster increase with leaf position in LER, leaf width and leaf area, higher relative leaf area expansion rates, and more biomass allocation to leaf sheaths and less to roots. No differences in leaf appearance rate were found amongst the species. CONCLUSIONS: Aegilops tauschii was the only wild species with rapid leaf expansion rates similar to those of wheat, and it achieved the highest photosynthetic rates, making it an interesting species for further study.     

Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol

Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol.     

Metamorphosis in the Cirripedia Rhizocephala and the homology of the kentrogon and trichogon

The Rhizocephala are considered to be monophyletic due to several synapomorphies in the ontogeny of the cndoparasitic phase. The various types of metamorphosis described in the Rhizocephala are discussed and compared to metamorphosis in the Cirripedia Thoracica and Acrothoracica. In males and females of the suborder Kentrogonida. the cyprid settles and metamorphoses into a new instar, in males the trichogen and in females the infective kentrogon. The kentrogon goes through yet another. incomplete moult associated with the development of the stylet. Within the three kentrogonidan families. the Iernaeodiscid-peltogastrid type of kentrogon differs from the sacculinid type in the mode of attachment to the host. in the complexity of internal anatomy. in the position and penetration of the stylet, and in whether or not the cyprid carapace must be shed prior to penetration of the stylet. In the Akentrogonida metamorphosis never results in a new instar. Where observed (Clistosaccidae and Thompsoniidae). both male and female cyprids settle and penetrate into their substrate (female parasite or new host) with one of the antennules. Using the antennule as a syringe. male cyprids inject spermatogonia while female cyprids injects embryonic cells developing into an endoparasite. By comparison with metamorphosis in the Cirripedia Thoracica and Acrothoracica it is concluded that the presence of a metamorphic moult leading to a post-cyprid instar is plesiomorphic and that the trichogon and kentrogon are homologous with the first metamorphosed juvenile in these outgroups. The abbreviated ontogeny in the Akentrogonida without metamorphic moult and post-cyprid larval instars is considered apomorphic. This contradicts the long-held supposition that the Akentrogonida are the most‘primitive’Rhizocephala and dovetails with new information that this suborder contains many advanced traits. Within the Kentrogonida. the lernacodiseid-peltogastrid type of kentrogon is considered more plesiomorphic than the sacculinid type, which resembles the clistosaccidthompsoniid type in having the antennules involved in the penetration process. The homologization of the kentrogon with a juvenile barnacle indicates that presence of a kentrogon is plesiomorphic within the Rhizocephala and that the Kentrogonida is paraphyletic.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Reduction of Oxidative Stress in Chronic Kidney Disease Does Not Increase Circulating α-Klotho Concentrations

The CKD-associated decline in soluble α-Klotho levels is considered detrimental. Some in vitro and in vivo animal studies have shown that anti-oxidant therapy can upregulate the expression of α-Klotho in the kidney. We examined the effect of anti-oxidant therapy on α-Klotho concentrations in a clinical cohort with mild tot moderate chronic kidney disease (CKD). We performed a post-hoc analysis of a prospective randomized trial involving 62 patients with mild to moderate CKD (the ATIC study), all using an angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB) for 12 months. On top of that, the intervention group received anti-oxidative therapy consisting of the combination of pravastatin (40 mg/d) and vitamin E (α-tocopherol acetate, 300 mg/d) while the placebo was not treated with anti-oxidants. α-Klotho concentrations were measured at baseline and after 12 months of anti-oxidant therapy. Data were analysed using T-tests and Generalized Estimating Equations, adjusting for potential confounders such as vitamin D, parathyroid hormone, fibroblast-growth-factor 23 (FGF23) and eGFR. The cohort existed of 62 patients with an eGFR (MDRD) of 35 ± 14 ml/min/1.72m², 34 were male and mean age was 53.0 ± 12.5 years old. Anti-oxidative therapy did successfully reduce oxLDL and LDL concentrations (P <0.001). α-Klotho concentrations did not change in patients receiving either anti-oxidative therapy (476.9 ± 124.3 to 492.7 ± 126.3 pg/mL, P = 0.23) nor in those receiving placebo 483.2 ± 142.5 to 489.6 ± 120.3 pg/mL, P = 0.62). Changes in α-Klotho concentrations were not different between both groups (p = 0.62). No evidence was found that anti-oxidative therapy affected α-Klotho concentrations in patients with mild-moderate CKD.     

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.