Influence of the PPAR-gamma2 Pro12Ala and ACE I/D polymorphisms on insulin sensitivity and training effects in healthy offspring of type 2 diabetic subjects.

Aerobic endurance training improves insulin sensitivity, and is of great importance in the prevention and treatment of type 2 diabetes. The improvement in insulin sensitivity and cardiovascular function through exercise is highly variable among individuals, and is probably partly determined by genetic components. This study evaluated the peroxisome proliferation-activated receptor-gamma2 ( PPAR-gamma2) Pro12Ala polymorphism and the angiotensin converting enzyme ( ACE) I/D polymorphism with respect to any potential influence that these highly prevalent polymorphisms may impose on changes in insulin sensitivity and maximal aerobic capacity induced by exercise. Seventy-nine healthy first-degree relatives of type 2 diabetic patients were compared to a control group consisting of 54 subjects without any family history of type 2 diabetes. All subjects had a normal OGTT. The groups were comparable with respect to age (34 +/- 7 vs. 33 +/- 7 years), gender ((m/f) 43/36 vs. 30/24) and BMI (25.7 +/- 2.6 vs. 25.3 +/- 2.5 kg/m (2)); p (all) = NS. Furthermore, a subgroup of 29 offspring and 17 control subjects were engaged in a standardized training program lasting ten weeks. Insulin sensitivity (hyperinsulinemic euglycemic clamp technique) and VO (2)max (exhaustive exercise test) was assessed before and after the training period. We will demonstrate the allelic frequency of the Ala-allele of the Pro12Ala polymorphism to be lower in offspring to type 2 diabetic patients (13.3 %) compared to control subjects (21.3 %); p < 0.05. In offspring only, the Pro12Ala polymorphism of the PPAR-gamma2 gene appeared to enhance weight changes brought about by exercise (Deltaweight = - 0.3 +/- 1.4 kg vs. - 1.8 +/- 1.8 kg; p < 0.05; (Pro/Pro vs. Pro/Ala + Ala/Ala) - suggesting possible gene-environment or gene-gene interactions. The ACE I/D polymorphism was not of significant importance in determining the capability of responding to exercise in terms of improvement in insulin sensitivity or maximal aerobic capacity.     

Diversity of native and alien plant species on rubbish dumps: effects of dump age, environmental factors and toxicity

Abstract. The flora of 96 rubbish dumps consisting of organic, inorganic and industrial wastes was studied in the Czech Republic. Some dumps contained toxic substances (heavy metals, chlorethylenes, phenols, polychlorinated biphenyls, oil hydrocarbons and biogas). Statistically significant factors explaining the number and proportional representation of native plant species, archaeophytes (introduced before 1500) and neophytes (introduced later) were determined. In total, 588 species of vascular plants were recorded, with archaeophytes (133 species) over‐represented and native species (322 species) and neophytes (133 species) under‐represented compared to their proportions in the national flora. Minimum adequate models were used to determine the effects of several factors on species numbers and proportions, independent of other factors. Dump area, human density in the region and altitude (non‐significant only in archaeophytes) were correlated positively with species numbers. Dump age, expressed as time since dump establishment, interacted with the dump toxicity; species numbers increased with dump age on non‐toxic dumps, whereas on toxic dumps no increase in numbers was noted. For neophytes, dump toxicity also interacted with human density; the increase in numbers of neophytes with human density is more pronounced on toxic than on non‐toxic dumps. The variables measured failed to explain observed differences in proportional representation of native species, archaeophytes and neophytes. This suggests that the occurrence of species growing in such extreme habitats is driven overwhelmingly by factors such as anthropogenic disturbance. A possible explanation for the positive effect of altitude on species numbers on dumps is that the effect of heating of the deposited substrate by microbiological processes, documented by previous studies, overrides the effect of altitude which was shown repeatedly to have a negative effect on species richness. Neophyte distribution is driven by an interplay of factors distinct from those influencing the distribution of native species, namely toxicity and human density (the latter we interpret as a surrogate for propagule pressure). Their distribution on studied dumps is more restricted than that of native taxa and archaeophytes, and they are more limited by toxic substrata; more intensive propagule pressure is required for their establishment at dumps with higher toxicity levels.     

Cell-based optimization of novel benzamides as potential antimalarial leads

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.     

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.