Effects of density-dependent inhibition of growth on phospholipid metabolism in monolayer cultures of animal cells.

The onset of density-dependent inhibition of growth is correlated with a change in the synthesis of the major phospholipids of either BHK-21 (hamster) and 3T3 (mouse) cells. The change consists of inhibition of phosphatidylcholine synthesis while the synthesis of phosphatidylethanolamine remains constant or increases. The incorporation of radioactive precursors reflects turnover rates, since no significant differences were observed in phospholipid composition. No major differences in fatty acyl chains of these same phospholipids were observed as a function of growth rate despite considerable individuality of relative patterns of polar lipids in both cell lines. No other density-dependent differences in phospholipid metabolism were observed which were common to both cell lines. These results suggest that major aspects of growth regulation may be metabolic rather than compositional and that regulation of the enzymes of lipid metabolism may be useful probes of effector mechanisms in growth control.     

Functional beta cell regeneration in the islets of pancreas in alloxan induced diabetic rats by (−)-epicatechin

(?)-Epicatechin (1), a naturally occuring flavonoid compound was found to have reversed the diabetogenic action of alloxan in albino rats (2). (?)-Epicatechin administration in doses of 30 mg/kg (i.p) twice daily for 4–5 days in alloxan induced (150 mg/kg, i.p.) diabetic albino rats (either sex), has brought down the high blood sugar levels to normal values. Concurrent histological studies of the pancreas of these animals showed regeneration of the beta-cell population of the islets which were earlier necrosed by alloxan. Immunoreactive insulin (IRI) studies showed that the regenerated beta-cells of the islets of pancreas are functional in nature.     

Effect of acyclic polyisoprenoids on the biosynthesis of mannose-labeled glycolipids in rat liver microsomes

The effect of the acyclic polyisoprenoid (C₂₀); geranylgeranyl-acetone (GGA) and its derivatives on mannolipid formation from GDP-mannose in rat liver microsomes was studied. Remarkable increases in mannolipid biosynthesis (Rf 0.24 and/or Rf 0.50) were observed by $\text{in vitro}$ additions of GGA, its hydroxylated compound (GGA-OH) and phosphorylated material (GGA-OP). These results strongly suggest that GGA-OP is involved in mannolipid formation (Rf 0.24) as an acceptor, in analogy to retinylphosphate. On the other hand, the mechanism for increased formation of an endogenous mannolipid (Rf 0.50) by GGA and GGA-OH is attributable to the enhancement of mannosyltransferase activity.     

Prevention by bombesin of cold-restraint stress induced hemorrhagic lesions in rats

Several neuropeptides, injected intraventricularly (ivt), were assessed for their effects on cold-restraint-induced hypothermia and hemorrhagic gastric lesions in 24 hr fasted rats. Bombesin (5-1 μg) further enhanced the drop in body temperature following stress and markedly prevented the gastric erosions in a dose-dependent fashion (5-0.1 μg). β-endorphin exerted a similar effect, but only at the 5 μg dose level. Other peptides (neurotensin, substance P, somatostatin and TRH: 5 μg) did not influence susceptibility to the gastric mucosal damage. Somatostatin and TRH reduced the hypothermic effect of stress. Bombesin is 250 times less potent when injected systemically than ivt and its actions are not reversed by nalaxone. The prevention of gastric erosions by bombesin could initially involve a central mechanism of action, independent of opiate receptors and possibly related to the sustained and marked hyperglycemia observed in bombesin treated rats exposed to stress.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Relationship between epitope density and immunoprecipitation of multivalent antigens by bivalent antibody: immunoprecipitation of adenylylated glutamine synthetase by anti-AMP antibodies

Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS₁₂^?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS₆^?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of ¹²⁵I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS₆^? or GS₁₂^? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS₁₂^?, whereas GS₆^? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS₆^?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS₁₂^?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS₆^? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS₆^? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS^?₁₂ by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS₆^? with anti-AMP antibodies is greater at 30 °C than at 4 °C.     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

Role of silicon in diatom metabolism : X. Polypeptide labelling patterns during the cell cycle, silicate starvation and recovery in Cylindrotheca fusiformis

The soluble polypeptides from Cylindrotheca fusiformis were labelled with [³⁵S]O₄²⁻ and resolved by two-dimensional gel electrophoresis. More than 600 polypeptides were detected upon a 26-day exposure to X-ray film. Analysis of the labelling pattern during the cell cycle show that labelling of at least 208 polypeptides changes; the majority, however, remain unchanged. Most of the changes occur in the beginning of the cell cycle and typically involve increases; those occurring in the second half of the cycle typically involve decreases. Light or its absence affects apparent protein turnover and the labelling rates of several polypeptides. Polypeptide labelling during the cell cycle was used as a reference to analyse the effect of silicate deprivation on diatom metabolism. In the absence of silicate, protein turnover increases: however, the addition of silicate counteracts but does not fully reverse this change. Silicate starvation affects the program of synthesis for several polypeptides, but in general the program of polypeptide labelling continues up to the S phase of the cell cycle. Addition of silicate to silicate-starved cells causes the appearance of four hitherto undetected polypeptides.     

Cell-to-cell ionic communication stimulated by 20-hydroxyecdysone occurs in the absence of protein synthesis and gap junction growth

When the larval epidermis of the beetle Tenebrio molitor is exposed to 20-hydroxyecdysone for 18–24 hr in vitro, the intercellular resistance to ion movement drops by about 30%. This drop in intercellular resistance is prevented when epidermal RNA synthesis is inhibited. It does not, however, appear to depend on de novo protein synthesis. Cycloheximide and emetine do not prevent the ecdysteroid from elevating junctional conductance. These drugs may even synergize with, or mimic, the action of the hormone on ionic coupling. A thin-section analysis of junctional ultrastructure showed that the absolute amount of gap junction at the epidermal cell interfaces does not alter after exposure to either 20-hydroxyecdysone or inhibitors of protein synthesis. These findings suggest that the low-resistance pathways in the beetle epidermis are not maximally conductive in the unstimulated state.     

Methylxanthine inhibition of neurosecretion in the brain and corpus cardiacum of Cecropia

The fine-structure of the median neurosecretory cells and corpora cardiaca of the Cecropia silkmoth during the first 7 days after transfer from cold conditions to room temperature was compared to that of similar animals whose development was arrested with aminophylline. The major difference observed was the failure of the intrinsic secretory cells of the corpus cardiacum to degenerate in the arrested animals. This failure to degenerate coincides with the expected period of brain hormone release. After long periods of arrest, the medial neurosecretory cells and their axons became distended with neurosecretory granules. The significance of these observations in the initiation of adult development is discussed.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

The regional metabolism of 5-hydroxytryptamine in mouse brain in vitro.

The relative rates of formation of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) from exogenous 5-hydroxytryptamine, showed regional variations when examined in homogenates of seven separate areas of mouse brain. 5-HTOL production was highest in the cerebellum, and lowest in the corpus striatum, whereas the production of 5-HIAA was greatest in the hypothalamus. Addition of NADPH was shown to increase the formation of the alcohol catabolite in whole brain homogenates. The production of 5-HTOL decreased in the brain homogenates of mice which had previously been injected with phenytoin sodium or oxypertine, with the latter also causing a fall in overall 5-HT metabolism.     

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

The dissociation of insulin from human insulin antibodies

The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k₁ = 2·1^?3 min^?1 and a fast (k₂ = 4·10^?2 min^?1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k₁ and k₂ values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6–850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achivement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k₁. The traditional concept that equilibrium is established at maximum binding requires further examination.     

Correlation of biochemical and morphological changes induced by chemical injury to the lung

Comparison has been made of injury to the rat pulmonary alveolar parenchyma evoked by intravenous injection of N-nitrosomethylurethane, intratracheal instillation of 3-methylcholanthrene or repeated inhalation for up to 15 days of carbon tetrachloride, trichloroethylene or gasoline vapour. Biochemical analyses, including assessment of rates of RNA and DNA synthesis and secretion of pulmonary surfactant, were correlated with morphological changes determined by electron microscopy. Single doses of N-nitrosomethylurethane or 3-methylcholanthrene inhibited incorporation of [14C] orotate into lung RNA 1--3 days after treatment. Daily exposure for 30 min to carbon tetrachloride or trichloroethylene vapour caused less marked reduction in orotate incorporation. Ultrastructural examination revealed that 3-methylcholanthrene toxicity was characterised by cytoplasmic change including disruption of surfactant lamellaie of Type 2 pneumocytes and variable degenerative changes Type 1 pneumocytes. Eight to ten days after treatment, the morphological evidence of hypertrophy/hyperplasia and transformation of Type 2 pneumocytes correlated well with biochemical evidence of stimulated incorporation of [3H]thymidine. Inhalation of carbon tetrachloride or trichloroethylene vapour produced milder responses including occasional degenerative changes in Type 1 pneumocytes, reduced numbers of surfactant lamellae in Type 2 pneumocytes and no change in [3H]thymidine incorporation. In contrast to the gradation of injury produced by the various chemicals, all procedures caused a marked and reproducible reduction in secretion of pulmonary surfactant as determined by endobronchial lavage. Following solvent inhalation, reduced recovery of surfactant was detected within 5 days of repeated exposure and thereafter no further change in this depressed level resulted from continued exposure for a further 10 days. The data are discussed in terms of a generalised pattern of response by pulmonary alveolar tissue to chemical injury and the apparent sensitivity of surfactant secretion as an indicator of damage to the lung.     

Uterine growth responses of the mature castrate rat to estradiol-17B

To examine estrogen-stimulated uterine growth we have monitored changes in uterine DNA synthesis, ornithine decarboxylase (ODC) activity and protein content as well as luminal epithelial (LE) cell mitotic index and ultrastructural changes. We have utilized this model to examine castrate mature rat uterine growth as a function of time between 18 and 40 hours following a single injection of 25.0 ug of estradiol-17B. LE cell mitotic index and protein content increases were maximally elevated as early as 18 hours postinjection while uterine ODC activity was maximal at 28 hours; uterine DNA synthesis increases continued throughout the experiment. In addition, the infusion of either 1 or 2 ug E2 plus progesterone over a 24 hour period, stimulated elevated ODC activity under both treatment regimens and LE cell mitotic index which was inversely related to E2 dose.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.     

Response of cell surface glycosyl transferases to dibutyryl adenosine-3',5' cyclic monophosphate in virus-transformed and normal cells.

Galactosyl and sialyl transferases in the plasma membrane of SV40-transformed mouse cells were inhibited by 0.5 mM dibutyryl adenosine-3′,5′ cyclic monophosphate (db-cAMP) while those of normal cells did not respond to this compound. The differential effects of dibutyryl adenosine-3′,5′ cyclic monophosphate on the membrane-bound glycosyl transferases were observed both in isolated plasma membrane and in intact cell membrane. It is suggested that some of the morphological restorations of normal cell characteristics during reverse transformation are partly due to the direct effect of this compound on the cell membrane.     

Acid- and temperature-induced conformational changes in human chorionic gonadotropin and the mechanism of subunit dissociation

Human chorionic gonadotropin undergoes a conformational transition in acid which at 4 °C is characterized by: (i) a reversible increase in the polarization of tyrosyl fluorescence, P, with a midpoint at pH 5, (ii) a slight decrease in the elution volume on Sephadex G-100 at pH 3 relative to pH 7, (iii) a slight decrease in s_20,w. (iv) a small positive near uv difference spectrum (Δ? ~2%), and (v) the appearance of a positive CD feature at 235 nm. These observations are compatible with an acid-expanded form of the hormone in which the rotational freedom of one or more tyrosine residues is restricted and/or their proximity to potential quenching groups is altered. The increased value of P following acidification is stable at temperatures below 10 °C, but at higher temperatures it decreases with time to an extent which is dependent on the temperature. A substantial portion of this decrease occurs before subunit dissociation can be detected, reflecting the occurrence of a thermal transition with a midpoint near 26 °C. A similar transition was observed at neutral pH with a midpoint near 22 °C. These results suggest the occurrence of at least two conformationally distinct forms of hCG which may be sequentially encountered prior to subunit dissociation in acid. The kinetics will be either biphasic or strictly first order, depending on the temperature at which the hormone is acidified.