Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

MATING SYSTEM AND ASYMMETRIC HYBRIDIZATION IN A MIXED STAND OF EUROPEAN OAKS

The sessile (Quercus petraea [Matt.] Liebl.) and pedunculate (Quercus robur L.) oaks are two closely related species having a wide sympatric distribution over Europe. Under natural conditions, they frequently form mixed forests, where hybridization is suspected to occur. In this paper, two different approaches have been applied to the study of the mating system and the interspecific gene flow in a mixed stand formed by the two species. The mating systems of both species have been studied separately by means of the mixed-mating model. The relative contribution of the parental species to the progenies have been estimated with two different methods. The first uses the admixture model. The second is an extension of the mixed-mating model and subdivides the outcrossing rate into intra- and interspecific components. The two species were almost completely outcrossing. This high level of outcrossing and interspecific gene flow could play an important role in the maintenance of the genetic diversity in these long-lived forest tree species. The contribution of the sessile oak to the pedunculate oak progenies varied from 17% to 48%. In contrast, ovules of sessile oak trees appear to be preferentially fertilized by other extreme sessile genotypes. We suggest that interspecific and directional gene flow was responsible for such patterns. Pedunculate oak is considered as a pioneer species and is progressively replaced by sessile oak. Our present findings add a further genetic component to this succession scheme, suggesting that unidirectional gene flow reinforces succession between the two species.     

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.     

Altitudinal zonation of montane Quercus forests along two transects in Chirripó National Park,Costa Rica

Abiotic and vegetation data were collected along two altitudinal transects through mature montane Quercus forests on the Pacific and Atlantic slopes of Costa Rica's Chirripó Massif. Between 2000 and 3200 m asl twenty-four 0.05 ha forest plots were selected at altitudinal intervals of 100 m, and eight soil profiles were described at intervals of 200 m. A TWINSPAN classification aided in the determination of eight zonal forest communities on the basis of their floristic composition. They are grouped in two sets of four: (i) the palm-rich lauraceous-fagaceous Lower Montane Mollinedia-Quercus Forests (2000–2600 m asl) and (ii) the bamboo-rich myrsinaceous-fagaceous Upper Montane Schefflera-Quercus Forests (2500–3200 m asl), respectively. Vegetation changes seem correlated with two major climatic gradients: (i) a temperature gradient (altitude), and (ii) a moisture gradient (wet Atlantic vs. moist Pacific slope). Most soils are Andepts, and residual, colluvial or derived from volcanic material. Humus layers are thicker on the wetter Atlantic slope. A total of 431 vascular plant species consisted of 86 pteridophytes, 1 gymnosperm, 296 dicots and 48 monocots. Species richness, canopy height and stem diameter decrease with increasing altitude, while the canopy surface becomes more flattend. A comparison with other studies shows that Chirripó's montane Quercus forests fit within the environmental ranges known from altitudinal zonations elsewhere in the Tropics.Abbreviations asl above sea level - dbh diameter at breast height - LM Lower Montane - Mt. Mountain - TWINSPAN two way indicator species analysis - UM Upper Montane - VU code referring to soil profiles as presented in Van Uffelen (1991) This paper is dedicated to the memory of Alwyn H. Gentry, an outstanding and inspiring tropical botanist who tragically died in a plane crash in the mountains of Ecuador on August 3 1993, when surveying possible boundaries for a new tropical cloud forest reserve.     

The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that theenvM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of anenvM mutant ofEscherichia coli. Ultimately,envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinantE. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.     

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes

An approximately 29-kD protein was purified from the membrane fraction of wheat (Triticum aestivum cv Dganit) mitochondria by the utilization of standard liquid chromatography techniques. The protein, designated MmP29 for mitochondrial membrane protein having a molecular mass of approximately 29 kD, exhibited cationic properties in a buffering solution, adjusted to pH 7.5. This positive charge enabled its passage through a diethylaminoethyl column, without interaction with the positively charged matrix. Subsequently, this protein was separated from the remaining polypeptides by a preferential elution from a hydroxylapatite/celite mixed column. Reconstituted liposomes containing this protein were characterized as being permeable to 8-amino-naphthalene 1,3,6-trisulfonic acid disodium salt (Mr 445) but non-permeable to dextran fluorescein (Mr 40,000). Additionally, MmP29 was inserted into planar phospholipid membranes, and anion-selective, voltage-dependent channels were demonstrated. All of the MmP29 properties mentioned highly resemble voltagedependent, anion-selective channel (VDAC) proteins, suggesting that MmP29 is the mitochondrial outer membrane VDAC protein of wheat.