Organization and chromosomal localization of the human interleukin 5 receptor alpha-chain gene.

The gene for the hIL-5R alpha subunit, which is present in a single copy in the human genome, has been analysed in detail. It is located on chromosome 3 in the region 3p26. The gene organization reflects its relationship to the cytokine/haematopoietin receptor superfamily. Three introns are located in the 5' untranslated region. The subsequent exons determine the functional domains of the hIL-5R alpha protein: the signal peptide, three fibronectin type III-like (FN-like) modules, each built up by two exons, the membrane anchor and two exons forming the cytoplasmic tail, the first of which contains the proline cluster region. In addition, a specific exon generating a soluble isoform is located before the membrane anchor exon. This specific exon contains an in frame TAA stop codon, followed by a polyadenylation signal. Hence, a normal splicing event leads to a soluble IL-5R alpha variant, whereas alternative splicing is required for cell membrane anchoring. A second area of alternative splicing is found in the 5' leader sequence, and possibly relates to the presence of short open reading frames preceding the main ATG. All intron-exon junctions meet the GT-AG rule. The gene structures of all cytokine/haematopoietin receptors documented so far have also been compared with respect to intron phasing. This shows that all introns between the FN-III-like modules are of the +1 type, but in addition, splice sites within the Cys-module and WS-WS-module are invariably of the +2 and 0 type, respectively.     

Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex

The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Nests of subterranean fungus-growing termites (Isoptera, Macrotermitinae) as nutrient patches for grasses in savannah ecosystems

Savannah are ecosystems in which mineral nitrogen is considered as a limiting factor for plant productivity. They are heterogeneous and spatially structured in patches, or islands, where mineral nitrogen content is concentrated. Among the soil macrofauna, termites of the Macrotermitinae subfamily are major determinants of soil heterogeneity through the biogenic underground nest structures (fungus‐comb chambers) they produce. To study the role of the heterogeneity created by termites on Pennisetum pedicellatum, an herbaceous grass species was grown in greenhouse. This was carried out using an homogeneous soil poor in mineral nitrogen, and an heterogeneous soil with patch, made of (i) Ancistrotermes cavithorax fungus‐comb chamber wall and (ii) soil with the same mineral nitrogen content as the termite handled soil. Plants exhibited a better growth on patch of termite‐modified soil whereas no significant differences were shown with the supply of mineral nitrogen. The presence of fungus‐comb chamber wall material resulted in an increase of fine root biomass and root/shoot ratio. We conclude that termites, through their building activities, may create nutrient patches available to grasses. Concurrently, our data illustrate that the higher mineral nitrogen content in termite‐built structures is not the only factor responsible for plant growth.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Expression of human hormone-sensitive lipase in white adipose tissue of transgenic mice increases lipase activity but does not enhance in vitro lipolysis

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters (CEs). The enzyme is highly expressed in adipose tissues (ATs), where it is thought to play an important role in fat mobilization. The purpose of the present work was to study the effect of a physiological increase of HSL expression in vivo. Transgenic mice were produced with a 21 kb human genomic fragment encompassing the exons encoding the adipocyte form of HSL. hHSL mRNA was expressed at 3-fold higher levels than murine HSL mRNA in white adipocytes. Transgene expression was also observed in brown adipose tissue (BAT) and skeletal muscle. The human protein was detected in ATs of transgenic (Tg) mice. The hydrolytic activities against triacylglycerol (TG), diacylglycerol (DG) analog, and CE were increased in transgenic mouse AT. However, cAMP-inducible adipocyte lipolysis was lower in transgenic animals. In the B6CBA genetic background, transgenic mice up to 14 weeks of age showed lower body weight and fat mass. The phenotype was not observed in older animals and in mice fed a high-fat diet (HFD). In the OF1 genetic background, there was no difference in fat mass of mice fed ad libitum. However, transgenic mice became leaner than their wild-type (WT) littermates after a 4 day calorie restriction. The data show that overexpression of HSL, despite increased lipase activity, does not lead to enhanced lipolysis.