4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.     

Modelling Metal Complexes of Calixarene Esters and Phosphine Oxides Using Molecular Mechanics

This paper focuses on the molecular modelling of a number of calixarene ester and phosphine oxide metal ion complexes. Monte Carlo conformational searches, in conjunction with the Merck Molecular Force Field, were carried out using Spartan SGI Version 5.0.1. running on Silicon Graphics O2 workstations. In the case of the calix[4]arene tetraesters, the optimised models strongly suggest that the selectivity of these ligands is strongly related to the eight-fold nature of the coordination with the Na⁺ ion, while coordination with the Li⁺ ion, for example, is merely three-fold. This feature of eight-fold coordination is also observed in the models of the complexes formed by the calix[4]arene tetraphosphine oxides with calcium. However, whereas the eight-fold coordination is unique to the model of the TPOL:Ca²⁺ complex among the ions modelled, this mode of coordination occurs for TPOS with sodium and potassium, in addition to calcium. This concurs with the observation that calcium selectivity is obtained with ion selective electrodes based on TPOL but not TPOS. Though the cavity in the calix[5]arenes PPOL and PPOLx and the calix[6]arene HPOL, in their uncomplexed form, are much larger than that of the corresponding calix[4]arenes, the pattern of selectivity is the same – the ligands are selective for calcium. The models of the complexes of these larger calixarenes, such as PPOL:Ca²⁺, strongly suggest that the reason for this similarity is that four of the available phosphine oxide groups complex with the calcium ion, and the others are forced away from the cavity region for steric reasons. The resulting eight-fold coordination, is therefore, similar to that of the calix[4]arenes studied.Electronic Supplementary Material available.     

Apolipoprotein-E dependent role for the FAS receptor in early onset Alzheimer's disease: finding of a positive association for a polymorphism in the TNFRSF6 gene

The TNFRSF6 gene encodes FAS, a cell-surface receptor involved in apoptosis initiation. Elevated levels of FAS have been reported in the brains of Alzheimer's disease (AD) patients. We have tested a G/A polymorphism at position -670 in the TNFRSF6 gene for association with non-familial, early onset Alzheimer's disease (EOAD) by using dynamic allele-specific hybridization. In an initial set of Scottish EOAD cases (n=78) and controls (n=152), we found that, for individuals carrying one or two APOE4 alleles, the homozygous GG-genotype was enriched in the patients (26.7% versus 10.9% in controls). A second study was conducted on an independent set of Scottish individuals (87 EOAD, 358 controls). In this material, the TNFRSF6 GG-genotype frequency was elevated in patients regardless of APOE4 status (28.7% versus 15.1%) and was even more enriched in APOE4 carriers (35.9% versus 15.3%). A combination of the two sample sets (165 cases, 510 controls) gave a significant disease association for the TNFRSF6 GG-genotype that was irrespective of APOE4 (P=0.0020) and that was almost completely attributable to the enrichment present within the set of APOE4 carriers (P=0.0016). This represents an odds ratio of 8.71 for GG-homozygotes carrying at least one APOE4 allele compared with other TNFRSF6 genotypes in APOE4 non-carriers. The TNFRSF6 variation was further explored in Scottish late-onset Alzheimer's disease (n=159) but no associations were found. These results imply that TNFRSF6, in interaction with APOE4, is a genetic risk factor for sporadic EOAD. Hence, the AD risk contributed by APOE4 could be mechanistically related to a pathway in common with FAS-mediated apoptosis.     

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.     

Molecular changes in the d-bifunctional protein cDNA sequence in australasian patients belonging to the bifunctional protein complementation group

The cDNA sequence for the human d-bifunctional protein (D-BP: 17β-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209–537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3–349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1–302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the β-oxidation defect in patients belonging to the BP CG.     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Reproductive burden, locomotor performance, and the cost of reproduction in free ranging lizards

Abstract. A reduction in the locomotor capacity of gravid females is considered to be a cost of reproduction if it leads to an increased risk of mortality. In this study, we measured the change in endurance between gravid and postgravid female side-blotched lizards ( Uta stansburiana ) as a test of the cost of reproduction. We also altered reproductive investment in some females by direct ovarian manipulation (yolkectomy), which decreased reproductive burden by 30%. Regardless of experimental treatment, all females had lower endurance when gravid. Endurance was 28% lower in gravid females from the yolkectomy treatment and 31% lower in the unmanipulated females relative to postoviposition females. The experimental reduction in clutch mass resulted in a 21% increase in endurance of gravid yolkectomy females relative to control females. Postovipositional endurance was significantly higher in the yolkectomized females than unmanipulated females, which suggests that the cost of reproduction carries over to postoviposition performance. Unmanipulated females exhibited a significant negative association between endurance and size-specific burden. Endurance was not correlated with clutch size or size-specific burden in the yolkectomy females. Survivorship to the second clutch was higher in the yolkectomy females. The results from a logistic regression showed the probability of survival to the second clutch was significantly and positively associated with endurance after controlling for the effects of treatment. Our analyses demonstrated that the decrement in performance associated with current reproductive investment represents a cost of reproduction expressed as diminished locomotor performance and lowered survivorship to the next clutch.     

Phenol oxidising enzymes in the grain aphid’s saliva

Sucrose-agarose gels and sucrose liquid diets were used to study the phenol oxidising enzymes in the salivary secretions of the grain aphid, Sitobion avenae (Fabricius). Activity indicating the presence of two oxidoreductases, polyphenol oxidase (PPO) and peroxidase (Px), was found. Both enzymes were present in the aphids stylet sheath (gelling saliva) but only polyphenol oxidase activity was found in the halos around sheaths and thus in watery saliva. Electrical penetration graphs (EPG) revealed that the secretion of these enzymes into the gels, by an individual aphid, was associated with its probing activity observed during penetration of the epidermal and mesophyll tissues. The grain aphids PPO, secreted in its saliva reacted with a range of phenolic compounds. As most of these phenolics occur naturally in cereals, the grain aphid could modify its host-plants phenolic composition. The importance of the grain aphids polyphenol oxidase and peroxidase in detoxifying cereal phenolics is discussed.     

Endothelin-converting enzyme in human tissues

Our aim was to determine whether the expression of endothelin-converting enzyme in human tissues would correlate with the distribution of its substrate, big endothelin-1, and its product, the mature peptide. Site-directed antisera raised against the conserved C-terminus of the mammalian enzyme were used to measure the immunoreactive enzyme in microsomal fractions prepared from tissue homogenates and to localize staining to the endothelial cells lining large conduit and smaller resistance vessels within cardiac, adrenal, respiratory and brain tissue. The activity of endothelin-converting enzyme was measured and characterized in isolated endothelial cells. This pattern of staining in the vascular endothelium paralleled that of mature endothelin and big endothelin-1, and these peptides were detectable by radioimmunoassay in all tissues examined. Immunoreactive endothelin-converting enzyme localized to other cell types, including bronchial epithelial cells, and to fibres within the glial limitans, neuronal processes and cell bodies of the cerebral cortex. Although perivascular astrocytes in the subcortical white matter displayed intense endothelin-converting enzyme-like immunoreactivity, endothelin staining was not detected. The results suggest that endothelin-converting enzyme has a ubiquitous distribution within the human vascular endothelium and is positioned to catalyse the conversion of big endothelin-1 to the biologically active endothelin-1, which on release may contribute to the maintenance of basal tone in humans. Endothelin-converting enzyme localized to epithelial cells in peripheral tissues or astrocytes within the brain may be upregulated in pathophysiological conditions in which endothelin levels are increased and could represent a further target for therapeutic intervention by enzyme inhibitors. © 1998 Chapman & Hall