Mannose, glucosamine and inositol monophosphate inhibit the effects of insulin on lipogenesis. Further evidence for a role for inositol phosphate-oligosaccharides in insulin action.

The mechanism of insulin signalling is not yet understood in detail. Recently, a role for inositol phosphate (IP)-oligosaccharides as second messengers transmitting the insulin signal at the post-kinase level was proposed. To evaluate this hypothesis further, we studied whether IP-oligosaccharides isolated from 'haemodialysate' have insulin-like activity. We found that these compounds mimic, in a dose-dependent fashion, the following effects of insulin in adipocytes. (1) Lipogenesis. Incorporation of [3H]glucose into lipids (expressed in nmol/min per 10(6) cells): basal, 0.74 +/- 0.05; insulin (1 mu unit/ml), 4.43 +/- 0.21; IP-oligosaccharide (2 micrograms/ml), 4.07 +/- 0.19. (2) Inhibition of isoprenaline (isoproterenol) (1 microM)-stimulated cyclic AMP levels and lipolysis. Cyclic AMP (pmol/10(5) cells): basal 0.84 +/- 0.05; isoprenaline, 4.03 +/- 0.19; isoprenaline + insulin (200 mu units/ml), 2.06 +/- 0.7; isoprenaline + IP-oligosaccharides (2 micrograms/ml), 2.4 +/- 0.29. Inhibition of lipolysis (mumol of glycerol/mg of protein): isoprenaline (1 microM), 166 +/- 11; isoprenaline+insulin (150 mu units/ml), 53 +/- 3.5; isoprenaline+IP-oligosaccharides (2 micrograms/ml), 58 +/- 5. (3) Stimulation of 3-O-methylglucose transport; basal, 9 +/- 3%; insulin (1 mu unit/ml), 67 +/- 4%, IP-oligosaccharides (2 micrograms/ml), 54 +/- 2%. To identify the active molecules of the IP-oligosaccharide fraction, competition experiments were performed. IP-oligosaccharide effects on lipogenesis were blocked by inositol monophosphate, glucosamine and mannose. In contrast, these compounds did not inhibit IP-oligosaccharide effects on membrane-mediated functions (3-O-methylglucose transport, cyclic AMP levels, lipolysis). We also found that the effect of insulin on lipogenesis was blocked by mannose, glucosamine and inositol monophosphate, whereas the insulin effects on 3-O-methylglucose, cyclic AMP and lipolysis were unaffected. The following conclusions were reached. (1) IP-oligosaccharides mimic the major metabolic effects of insulin in adipocytes. This is consistent with the proposed role of IP-oligosaccharides as second messengers of certain insulin effects. (2) Mannose and glucosamine are functionally important sugar residues for the effect of IP-oligosaccharide on lipogenesis. (3) The observation that mannose, inositol monophosphate and glucosamine block the action of insulin of on lipogenesis supports a role of mannose- and glucosamine-containing IP-oligosaccharides as second messengers for this insulin effect.     

Further evidence for an allosteric model for ribonuclease.

Evidence is presented from three experimental systems to support the allosteric model of Walker et al. (1975) (Biochem. J. 147, 425-433) which explains the substrate-concentration-dependent transition observed in the RNAase (ribonuclease)-catalysed hydrolysis of 2':3'-cyclic CMP (cytidine 2':3'-cyclic monophosphate). 1. Kinetic studies of the initial rate of hydrolysis of 2':3'-cyclic CMP show that the midpoint of the transition shifts to lower concentrations of 2':3'-cyclic CMP in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, 3'-UMP and Pi; 2'-CMP and 2'-UMP do not cause such a shift. 2. Trypsin-digestion studies show that a conformational change in RNAase to a form less susceptible to tryptic inactivation is induced in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, and 3'-UMP. 2'-CMP, 2'-AMP and 2'-UMP do not induce this conformational change. 3. Equilibrium-dialysis experiments demonstrate the multiple binding of molecules of 3'-CMP, 3'-AMP and 5'-AMP to a molecule of RNAase. 2'-CMP binds the ratio 1:1 over the analogue concentration range studied.     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

Further evidence for hyaluronidase activity of Treponema pallidum

The presence of hyaluronidase in preparations of Treponema pallidum was previously shown using acidified bovine serum albumin reactions and Ouchterlony immunodiffusion. To expand on these preliminary findings more sensitive techniques of viscometry, additional immunologic reactions, and altered capillary permeability were used to characterize treponemal-associated hyaluronidase. The pathogens T. pallidum and T. pertenue degraded hyaluronic acid, whereas the nonpathogens T. denticola and T. vincentii did not. As syphilitic infection progressed, hyaluronidase activity decreased; organisms harvested from 14-day testicular infections degraded hyaluronic acid less rapidly than organisms from 4-day infections. Uninfected rabbit testicular extract also exhibited significant enzyme activity. The neutralizing activity of immune sera was decreased by prior adsorption with bovine hyaluronidase, suggesting that some of the neutralizing factors are associated with this enzyme. Radioimmunoassay was used to quantitate antibodies to hyaluronidase in immune sera. Antihyaluronidase sera were isolated from rabbits immunized with bovine hyaluronidase. Treponema pallidum, as well as uninfected rabbit testicular extract, cross-reacted with these antisera. Immunofluorescence indicated that the hyaluronidase was uniformly distributed along the treponemal surface. As a final indicator of hyaluronidase activity, alterations in capillary permeability were detected 1 h after intradermal injection of T. pallidum.     

Further evidence for non-T-cell regulation of delayed hypersensitivity in the guinea pig.

The nature of the suppressor activity in the spleens of guinea pigs immunized with dinitrophenyl-bovine γ-globulin in Freund's incomplete adjuvant was investigated. An anti-T-cell serum was prepared in rabbits and, after extensive absorption, showed specific killing for T-lymphocytes. After treatment with this antiserum and complement, spleen cells from animals immunized with the antigen in Freund's complete adjuvant showed marked reduction in ability to transfer sensitivity to normal recipients. However, when immune spleen cells, treated in the same way, were transferred into antigen immunized animals which had been pretreated with cyclophosphamide, the suppressor activity was unaltered. These results confirm earlier impressions that the regulation of delayed hypersensitivity reactions in the guinea pig is normally mediated by non-T-cells.     

Further evidence for a possible role of trypsin-like activity in the adherence of Porphyromonas gingivalis.

The present study was undertaken to evaluate a possible correlation between the level of trypsin-like activity and the adherence properties of Porphyromonas gingivalis. It was demonstrated that strains with high cell-associated trypsin-like activity attach in higher numbers to human epithelial cells than strains with low levels of trypsin-like activity. To a lesser extent, the same tendency was also noted for the agglutination of human erythrocytes. The ability of P. gingivalis to attach to erythrocytes and epithelial cells was found to be affected by the presence of arginine and thiol protease inhibitors (leupeptin, p-chloromercuriphenylsulfonic acid). The inhibition profile was partially dependent on the age of the bacterial cells used in the adherence assay. It is suggested that adherence of mid-log P. gingivalis cells involves primarily trypsin-like proteases, whereas 2-day-old cells possess additional specific attachment mechanism(s).     

Further evidence for a flexible and highly expanded spheroidal model for mucus glycoproteins in solution.

The flexible and greatly expanded roughly spherical model for mucus glycoproteins proposed earlier, on the basis of hydrodynamic and n.m.r. data, is supported by new hydrodynamic results on a bronchial glycoprotein from a cystic-fibrosis patient. Furthermore, images from electron microscopy of this molecule and a lower-molecular-weight mucus glycoprotein (which closely resembles a glycopolypeptide) appear to be at least consistent with this model.     

Further evidence for Darwin's pangenesis.

Darwin's pangenesis, a developmental theory of heredity, has been largely thought to be wrong for more than a century. In this paper, further evidence for the inheritance of acquired characters and graft hybridization is provided. A striking similarity between mRNA and Darwin's so-called gemmule has been found by comparing their nature and function. I propose that once the term gemmule has been replaced by mRNA, Darwin's pangenesis will revive, indicating an important step in biology.     

Inositol trisphosphate regulation of photoreceptor membrane currents.

In previous studies elevation of intracellular Ca2+ was shown to cause prolonged reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the membrane of the isolated Hermissenda photoreceptor, the type B cell (Alkon et al., 1982b; Alkon and Sakakibara, 1985). Here we show that iontophoretic injection of inositol trisphosphate (IP3), but not inositol monophosphate, also caused prolonged reduction of IA and ICa2+-K+. IP3 injection also caused reduction of a light-induced K+ current (also ICa2+-K+) but did not affect the voltage-dependent Ca2+ current, ICa2+, or the light-induced inward current, INa+, of the type B cell. IP3 injection caused similar effects on the K+ currents of the other type of Hermissenda photoreceptor, the type A cell. INA+ of the type A cell, unlike that of the type B cell, was, however, markedly increased following IP3 injection. The differences of IP3 effects on the two types of photoreceptors may be related to differences in regulation of ionic currents by endogenous IP3 as reflected by clear differences (before injection) in the magnitude of IA, ICa2+-K+, and INa+ between the two cell types.     

Further evidence for the regulation of bacterial populations in soil by protozoa

After the addition to soil of large numbers of a cowpea Rhizobium strain, the population declined steadily until the numbers reached about 10⁷/g, and the protozoa rose to about 10⁴/g. When indigenous protozoa were suppressed by the addition of actidione to the soil, the density of the test rhizobium did not fall initially, but its abundance declined to about 10⁷/g when actidione-resistant protozoa arose in significant numbers. The addition to actidione-treated soil of an antibiotic-resistant strain of Paramecium led to a rapid decrease in the population of the rhizobium, the density reaching essentially the same value as in soil receiving neither the drug nor the paramecia. The same changes occurred with Xanthomonas campestris as test prey except that its numbers fell to about 10⁵/g of soil. These data provide further evidence for the key role of protozoa in controlling the abundance of populations of certain bacteria introduced into soil.     

Further evidence for the 1,25-dihydroxyvitamin D-like activity of Solanum malacoxylon

Administration of an aqueous extract of the calcinogenic plant $\text{Solanum}\text{malacoxylon}$ (S.m.) to vitamin D-deficient or strontium fed chicks produces significant plasma 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) activity within 6 hr. (via radioreceptor assay) and subsequently elicits the appearance of immunoreactive intestinal calcium binding protein. Studies of a purified aqueous extract of S.m. show that it does not compete effectively with radioactive 1,25-(OH)₂D₃ for binding to the sterol's intestinal receptor. However, treatment of the extract with β-glucosidase releases a biologically active substance which is soluble in organic solvents and efficiently competes with labeled sterol for the receptor. This factor migrates exactly with tritiated 1,25-(OH)₂D₃ on high resolution Celite liquid-liquid partition columns. Thus, S.m. contains a molecule very similar or identical to 1,25-(OH)₂D₃ which is combined with one or more carbohydrate moieties in the native plant. This glycoside is probably cleaved $\text{in}\text{vivo}$ before biological activity is attained.     

Further evidence for a predominantly maternal transmission of maturity-onset type diabetes.

A significantly increased maternal diabetes transmission was observed in young diabetics with maturity-onset type diabetes as compared to those with classical juvenile-onset type diabetes (P less than 0.001). With respect to paternal diabetes transmission, on the other hand, there was not found a similar difference between both types of diabetes mellitus.     

On ferritin heterogeneity. Further evidence for heteropolymers

Tissue ferritins from the horse, rat, and human consist of multiple isoferritins some of which are common to more than one tissue in the same individual. Subunit analyses indicate that the ferritins from all three species are similarly composed of only two types of subunit with an approximate Mr of 21,000 and 19,000, designated H and L. The relative amounts of these subunits vary progressively throughout the isoferritin spectrum. Amino acid analyses and tryptic peptide maps indicate that the H and L subunits have extensive sequence homologies and that both are species-specific. Both subunits have been identified as the primary products of apoferritin synthesis in a wheat germ lysate programmed by rat liver mRNA. These results substantiate our proposal (Adelman, T. G., Arosio, P., and Drysdale, J. W. (1975) Biochem. Biophys. Res. Commun. 63, 1056-1062) that tissue ferritins are not unique homopolymers but families of hybrid molecules consisting of different proportions of two subunit types.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Kinetic model of regulation of muscle protein activity.

The widely accepted steric model of calcium regulation of actin-myosin interactions in vertebrate muscles has to be completed to fit the kinetic data. It should be supposed that: (1) the thin filaments consist of functionally independent units, containing seven actin sites regulated by one troponin-tropomyosin complex; (2) actin sites become available for myosin heads only due to fluctuations of tropomyosin position; (3) binding of calcium to troponin results either in the shift of the tropomyosin equilibrium position or in the weakening of its interactions with actin strand so that the probability of effective fluctuations increases; (4) link formation between myosin head and some of the available actin site fixates the tropomyosin in such a position that the other six actin sites of the same functional unit become available for myosin too.The model gives linear kinetic scheme for the transitions of a functional unit between nine states (a “turned off” state, and eight “turned on” ones with different occupancy by myosin heads). The dependences of the apparent rate constants of actomyosin formation and dissociation upon the myosin head and substrate concentrations are obtained from the Lymn-Taylor scheme. The frequency of the actomyosin complexes dissociation is assumed to give the ATPase rate.The model fits the kinetic data on the ATP hydrolysis by myosin subfragment-1 with regulated or unregulated actin as a cofactor under various conditions. It shows a sharp dependence of activation upon the apparent affinity of the actin and myosin sites. Therefore, the model appears to be applicable to myosin controlled systems.     

Kinetic evidence for a two-step mechanism for the binding of chymotrypsin to alpha 1-proteinase inhibitor.

We have used the proflavin displacement method and a stopped-flow apparatus to measure the rate constant for the binding of 2 microM-chymotrypsin to 20-125 microM-alpha 1-proteinase inhibitor. The observed pseudo-first-order constant showed a hyperbolic dependence on alpha 1-proteinase inhibitor concentration, suggesting a reaction mechanism in which a fast pre-equilibrium (K = 0.19 mM) is followed by a first-order formation of the final complex (k = 252 s-1).