Two different protein kinase activities are associated with the insulin receptor.

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.     

Discrimination between groups of chromosomes and individual chromosomes in the normal human karyotype

The karyotypes of 200 normal individuals are examined in a study designed to investigate the extent to which the cytologist's classification of chromosomes into the A–G autosomal groups and identification of individual chromosomes are reflected in the values of measurements made on these chromosomes. The statistical technique of discriminant function analysis is employed to obtain linear functions of the chromosomal measurements which may be used to classify and identify chromosomes. The results of the study indicate that while chromosomes can be grouped with a reasonably high probability of success using measurements alone, identification of individual chromosomes is somewhat more difficult.     

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Interactions among serum vitamin D binding protein, monomeric actin, profilin, and profilactin

Human serum vitamin D binding protein (hDBP), a 58-kDa inter-alpha-globulin, is known to bind, monomeric actin (G-actin) in equimolar quantities. Using monoclonal and polyclonal anti-hDBP antibodies, hDBP, and radioiodinated actin, we developed a reliable saturation assay for actin bound to hDBP. By utilizing this assay, kinetic analysis, and ultracentrifugal sedimentation in sucrose gradients, these proteins' binding affinities (Kd = 10(-9) M) were demonstrated to be 10- to 100-fold greater than earlier estimates. At 4 degrees C, hDBP has an association rate constant of 2.2 x 10(4) M-1 s-1 and a rate of dissociation displaying a t1/2 of 22 h. This high affinity binding was largely unaffected by conditions favoring actin filament formation (1 mM MgCl2 and/or 50 mM KCl), by the range of pH from 6.8 to 8.6 or by temperatures from 4 to 37 degrees C. Compared with ATP-alpha-actin, a 2-fold decrease of binding affinity was observed for the nonmuscle isoactins (beta,gamma), ADP-G-alpha-actin, and N'-ethylmaleimide-modified G-alpha-actin. The 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 holo-sterol forms of hDBP bound actin in a manner indistinguishable from the apo-sterol hDBP. The common polymorphisms of hDBP (DBP1 slow, DBP1 fast, and DBP2) were shown to have an equal avidity for G-actin binding. Human platelet profilin competed with hDBP for binding to G-actin, but was 1000-fold less potent (Ki = 1.9 microM). When platelet profilactin was incubated with hDBP, profilin was liberated and hDBP-actin complexes formed. DNase I, which forms a triprotein complex with hDBP-G actin, did not alter the affinity of binding of actin by hDBP. The very high affinity binding observed, which was largely unaffected by the state of G-actin, pH, and ionic conditions, appears to support a constitutive role for plasma DBP in the sequestration of actin monomers, as well as actin from actin-profilin complexes, that are liberated during cell injury.     

Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.     

Bronchial circulation in pulmonary artery occlusion and reperfusion

Obstruction of pulmonary arterial blood flow results in minimal biochemical and/or morphological changes in the involved lung. If the lung is reperfused, a syndrome of leukopenia and lung edema occurs. We used the radiolabeled microsphere technique to measure the response of the bronchial circulation in rabbits to acute pulmonary artery occlusion (PAO) and to pulmonary artery reperfusion. We found that the bronchial blood flow (Qbr) decreased from a base line of 0.37 +/- 0.10 to 0.09 +/- 0.04 (SE) ml.min-1.g dry lung-1 (P less than or equal to 0.05) after 4 h of PAO. In a separate group of animals, Qbr 24 h after PAO remained low (0.20 +/- 0.07 ml.min-1.g dry lung-1, P = 0.06). Qbr during PAO was inversely correlated with the wet-to-dry ratio after reperfusion (r = -0.68, P = 0.06). Qbr did not change during 4 h of reperfusion. We speculate that a critical level of Qbr may be necessary during PAO to prevent ischemia/reperfusion injury from occurring.     

A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome.

We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required for origin activity in the chromosome, as assayed by two-dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3' to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3'-flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild-type 3'-flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3'-flanking sequence serves as a DNA unwinding element in the chromosome.     

cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.     

Serum concentration of prolactin, gastrin and glycocholic acid in patient with peptic ulcer treated with ranitidine

In 66 patients with peptic ulcer (11 with gastric ulcer, 55 with duodenal ulcer, 19 women, 47 men) the serum concentrations of prolactin, dehydrocholic acid and gastrin were determined. The studies were repeated after treatment with ranitidine: in 50 patients after three weeks and in 40 patients after another 30 days. During the first period ranitidine 2 x 150 mg was administered, while during the second period the dose was 1 x 150 mg. The results were compared with those obtained from 120 healthy subjects. Before starting the treatment prolactin levels were significantly higher than those in the control group. During the treatment a significant decrease of the levels was observed. Similar changes of prolactin concentrations were found in the group of 39 men with duodenal ulcer isolated from the studied patients, who were compared with a group of 50 healthy men. It was not found that the development of peptic ulcer and the treatment with ranitidine exerted and effect on the changes of gastrin and dehydrocholic acid concentrations.