Structural organization of the human insulin receptor ectodomain.

To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.     

Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin.

We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed.     

The anatomy, physiology, acoustics and perception of speech: essential elements in analysis of the evolution of human speech

Inferences on the evolution of human speech based on anatomical data must take into account its physiology, acoustics and perception. Human speech is generated by the supralaryngeal vocal tract (SVT) acting as an acoustic filter on noise sources generated by turbulent airflow and quasi-periodic phonation generated by the activity of the larynx. The formant frequencies, which are major determinants of phonetic quality, are the frequencies at which relative energy maxima will pass through the SVT filter. Neither the articulatory gestures of the tongue nor their acoustic consequences can be fractionated into oral and pharyngeal cavity components. Moreover, the acoustic cues that specify individual consonants and vowels are “encoded”, i.e., melded together. Formant frequency encoding makes human speech a vehicle for rapid vocal communication. Non-human primates lack the anatomy that enables modern humans to produce sounds that enhance this process, as well as the neural mechanisms necessary for the voluntary control of speech articulation. The specific claims of Duchin (1990) are discussed.     

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.     

Cultural conditions forAeromonas hydrophila affect the production of haemolysins with differing host specificities

Five strains ofAeromonas hydrophila were studied for production of haemolysin specific for erythrocytes of various animal species using three cultural methods. All the strains produced haemolysin for all the erythrocyte species when the organisms were cultured on blood agar.Using cellophane overlay method, all the strains produced haemolysin for fish erythrocytes and variable activity to mammalian erythrocytes. Only one strain produced haemolytic activity for various though not all of the erythrocyte species when grown in brain heart infusion broth.Data suggest thatA. hydrophila produces multiple haemolysins with specificities for erythrocytes of different animals. This was confirmed for trout and horse erythrocyte targeted haemolysins, by using iso-electric focussing separation and by measuring the effect of addition of ammonium sulphate to the growth medium.     

Growth factor requirements of organogenesis in serum-free metanephric organ culture

Summary In order to define humoral growth factors which may regulate mammalian renal development, the growth requirements of fetal metanephric organogenesis were studied in serum-free murine organ culture. Metanephric growth, determined by cell proliferation and protein content, and metanephric differentiation, determined morphometrically as epithelial glomerular formation, were compared and contrasted following 144 hours of organ culture incubation in basal medium, basal medium supplemented with 10% fetal bovine serum, and basal medium supplemented with various combinations of growth factors. The basal medium was composed of equal volumes of Dulbecco's modified Eagle's medium and Hams' F-12 medium. Five humoral growth factors were studied in the following concentrations: selenium, 6.8×10⁻⁹ M; insulin, 8.3×10⁻⁷ M; triiodothyronine, 2×10⁻⁹ M; transferrin, 6.2×10⁻⁸ M; and prostaglandin E₁, 7.1×10⁻⁸ M. Results showed that transferrin and prostaglandin E₁ were necessary for optimal growth in the system and that prostaglandin E₁ was necessary for maximal metanephric differentiation. Such data provide guidelines for the creation of serum-free medium for future fetal renal cell and tissue culture systems, and provide insight into the factors which may regulate normal and abnormal renal embryogenesis and the reparative processes of renal hyperplasia and hypertrophy which follow renal injury. A preliminary report of this work was presented at the Ninth International Congress of Nephrology, Los Angeles, June 1984. These studies were supported in part by Basil O'Connor Starter Research Grant 5-349 from the March of Dimes Birth Defects Foundation and New Investigator Research Award I-R23-AM34891-01 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases of the National Institutes of Health (Both to Dr. Avner). Editor's Statement The determination of effects of extracellular components on the introduction and maintenance of differentiation is an area for which serum-free culture techniques are particularly suited. The approaches described in this report utilize morphometric techniques to quantitate differentiation in serum-free fetal organ culture in addition to standard methodologies for assessing growth. The purely epithelial nature of the cultures used in these studies also provides some interesting advantages in the design of experiments aimed, at determining the importance of cell-cell interactions at various stages in the differentiative process. David W. Barnes     

Spatial and temporal relationships between cell death and tissue overgrowth in imaginal wing disks of the Drosophila mutant I(3)C43hs1

The temperature-sensitive mutant l(3)c43^hs1 is lethal at the restrictive temperature late in the last larval instar and has wing disks that show excessive growth when larvae are reared at 25°C. Such mutant disks give rise to defective wings showing duplications and deficiencies. Abnormal folding patterns are localized to the region between the wing pouch and the area where adepithelial cells are found; the disks retain an epithelial morphology. Apoptotic cell death is distributed throughout the wing disks without any obvious concentration of dead cells in a specific area. Cell death is seen as early as 12 hr after a shift to the restrictive temperature. Temperature shift experiments also show that cell death precedes the onset of overgrowth, but since the spatial distribution of death is not localized to the regions of abnormal folds, it is unlikely that cell death and overgrowth are causally related.     

Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 10³ g for 10 min (F1) and 10⁴ g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 10⁵ g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 m in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.