Treatment of childhood myopia with atropine eyedrops and bifocal spectacles.

BACKGROUND: Animal and human studies have suggested that muscarinic antagonists and bifocal spectacles may decrease the progression of myopia in children. The purpose of this study is to report the largest known series of patients treated simultaneously with bifocals and topical atropine. DESIGN: Retrospective, interventional, non-comparative case series. METHODS: 706 myopic children (296 boys and 410 girls, ages 6 to 16 years) were prescribed full cycloplegic spectacle corrections, with photochromic lenses and +2.25 diopter (D) reading adds in each eye. Both eyes were treated with atropine 1% drops once daily. OUTCOME MEASURES: Annual change in cycloplegic refractions of right eyes. Compliance with therapy was monitored by patient and parental report. RESULTS: 496 (70%) of the 706 patients reported full compliance with the treatment regimen, whereas the remaining 210 (30%) patients were partially compliant. The median interval of treatment was 3.62 years (range, 21 days-10.1 years). The mean rate of myopic progression was significantly less (P<0.001) in patients who were fully compliant with atropine therapy and bifocals (0.08 D/year) than in patients who were partially compliant with the treatment regimen (0.23 D/per year). No serious adverse effects were associated with atropine therapy. CONCLUSIONS: Full compliance with topical atropine therapy and bifocal spectacles was associated with decreased progression of myopia compared to partial compliance with treatment. For each of the treated groups, the mean rate of myopic progression was significantly less (P<0.05) than the mean annual rates of myopic progression published for the pediatric population.

Evidence for stimulation of adenylyl cyclase by an activated G(s) heterotrimer in cell membranes: an experimental method for controlling the G(s) subunit composition of cell membranes.

Heterotrimeric (alphabetagamma) G(s) mediates agonist-induced stimulation of adenylyl cyclase (AC). Cholera toxin (CTx) will ADP-ribosylate the alpha-subunit of G(s) (G(s)alpha). G(s)alpha-deficient cyc(-) membranes were "stripped" of Gbeta. When the stripped cyc(-) were incubated with G(s)alpha and/or Gbetagamma, each was incorporated into the membranes independently of the other. Both G(s)alpha and Gbetagamma had to be present in the membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate G(s)alpha, indicating that the membrane bound G(s) heterotrimer is a substrate for CTx, but the G(s)alpha subunit by itself is not. When G(s)alpha was completely and irreversibly activated with GTPgammaS and incorporated into stripped cyc(-), it was a poor substrate for CTx and a weak stimulator of AC unless Gbetagamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of G(s) heterotrimer that was formed in the membranes from GTPgammaS-activated G(s)alpha and Gbetagamma. These data suggest that AC is stimulated by an activated G(s) heterotrimer in cell membranes.

Pesticide inactivation of peanut glutamate dehydrogenase: biochemical basis of the enzyme's isomerization.

Glutamate dehydrogenase (GDH) isomerizes in response to pesticides and environmental chemicals, but the biochemical basis of the isomerization is not known. Clearer understanding of the isomerization would permit expansion of its utility in the diagnosis of the responses of plant tissues to challenged environments. Peanut plants were treated with different rates of Basagran (3-(1-methylethyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide), Bravo 720 (tetrachloroiso-phthalonitrile), and Sevin XLR Plus (1-naphthyl N-methylcarbamate). Free solution isoelectric focusing, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fractionated the peanut seed GDH to its constituent subunits and degradation polypeptides. After western transfer to nitrocellulose membrane, the GDH subunits and degradation polypeptides were immunodetected with anti-GDH. The pesticide treatments did not induce increased proteolytic activity, but induced about 50% degradation of the GDH, whereas the GDH of the control peanut suffered only about 25% degradation, thus showing that the degradation rate was about double the rate of de novo synthesis in the pesticide treatments. The heavy displacement of the GDH subunit equilibrium toward degradation explains the biochemical basis of the isomerization reaction.

[Laparoscopic anti-reflux surgery--report of experiences in Austria]. / Laparoskopische Antirefluxchirurgie--Erfahrungsbericht aus Osterreich.

In Austria neither the open nor the laparoscopic fundoplication can be described as a routine operation. The number of laparoscopic operations is increasing year by year, the indication for surgery is more determined by radiological than by functional diagnostic tools. In our own patients the complication rate in 196 laparoscopic operations is 6%, regarding symptoms of reflux and dysphagia. Dysphagia was found only in cases with 360 degrees fundoplication, whereas recurrent reflux occurred in 270 degrees hemifundoplications (Toupet). The mortality rate following laparoscopic fundoplications was zero, morbidity below 10%.

Prelymphomatous B cell hyperplasia in the bone marrow of interleukin-7 transgenic mice: precursor B cell dynamics, microenvironmental organization and osteolysis.

Transgenic mice carrying mouse interleukin-7 (IL-7) cDNA under the control of MHC class II (E alpha) promoter develop B lymphoid tumors. We have analyzed population dynamics of early precursor B cells and electron microscopic organization of bone marrow (BM) during the prelymphomatous phase. Immunofluorescence labeling of terminal deoxynucleotidyl transferase (TdT), B220 glycoprotein, and mu heavy chains have been used to quantitate three populations of pro-B cells lacking mu chains, cytoplasmic mu-bearing pre-B cells, and surface mu-bearing B lymphocytes. Proliferative activity was assayed by metaphase arrest. In BM of IL-7 transgenic mice, the number and proliferative activity of cells in each of the pro-B and pre-B cell populations were markedly increased. B lymphocytes increased to a lesser extent. The BM cavity was considerably expanded and cortical bone showed focal osteolysis. Immature lymphoid cells compressed the venous sinusoids and exuded through eroded bone. Apoptotic bodies, macrophages, and plasma cells were unusually prominent. B lymphocytes and cells of B precursor phenotype were also much increased in the spleen. These results demonstrate that overexpression of IL-7 causes excessive proliferation of a wide range of precursor B cells in BM. Such prolonged stimulation at early stages of B cell development, prone to genetic errors, may predispose to neoplasia. The bone resorption in these transgenic mice provides a model for bone lesions in BM malignancies.

Regulation of glycogen synthase activity in isolated rat adipocytes by levamisole.

The effect of levamisole on glycogen synthase activity in isolated adipocytes was studied. The addition of levamisole to these cells resulted in an acute concentration-dependent increase in glycogen synthase activity. In contrast, epinephrine, dibutyryl cyclic AMP (DcAMP) and cysteamine decreased glycogen synthase activity. The stimulatory effect of levamisole on the activity of the enzyme was not affected by the presence of epinephrine but was diminished when either DcAMP or cysteamine was present. The results of this study suggest that levamisole increases adipocyte glycogen synthase activity by a mechanism that can be reversed by the elevation of cAMP.

The effect of digitonin and altered thyroid status on palmitic acid oxidation by isolated rat liver mitochondria.

The effect of altered thyroid status and food-deprivation on palmitic acid oxidation in isolated rat liver mitochondria was studied in the absence and presence of digitonin. Mitochondria prepared from triiodothyronine-treated (hyperthyroid) and food-deprived rats metabolized palmitic acid at the same rate as the untreated controls (euthyroid). Mitochondria prepared from thyroidectomized (hypothyroid) rats metabolized palmitic acid at a rate lower than was that seen with mitochondria from euthyroid controls in either the fed or fasted state. Fasting had no effect on palmitic acid oxidation by mitochondria prepared from euthyroid rats but diminished the rates seen in both hyper- and hypothyroid states. Digitonin (0.04 mg/mg mitochondrial protein) increased the sensitivity of the rate of fatty acid oxidation to inhibition by alpha-bromopalmitic acid. The addition of digitonin to the incubation mixture resulted in two-fold increases in the rate of palmitic acid oxidation in all states. This study shows that the limitations imposed by hypothyroidism on fatty acid oxidation in intact liver are preserved in isolated mitochondria.

Regulation of pyruvate dehydrogenase activity in rat fat pads and isolated hepatocytes by levamisole.

The effect of levamisole on the catalytic activity of the pyruvate dehydrogenase (PDH) complex in rat adipose tissue segments and isolated hepatocytes was studied. Levamisole, an anthelmintic and immunotherapeutic agent with concentration-dependent oxidant/antioxidant properties, increased the catalytic activity of the pyruvate dehydrogenase complex in these systems. The activity of this enzyme complex may be regulated in part by protein-thiol modification. The treatment of rat adipose tissue segments and isolated hepatocytes with and without levamisole (0.1-3.0 mM) for 30 min resulted in a reversible increase in the catalytic activity of this enzyme complex. Levamisole at 1.5 mM and 2.0 mM were as potent as insulin (1.0 mU/ml) in adipose tissue and as dichloroacetate (2.0 mM) in isolated hepatocytes, respectively, in increasing the catalytic activity of the PDH complex. The stimulatory effects seen with levamisole were not accompanied by decreases in adenosine triphosphate (ATP) levels. The results of the present investigation suggest that the pyruvate dehydrogenase complex in adipose tissue and liver may be useful models for studying the mechanism of action of levamisole.

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen.

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.

Interaction between insulin and thyroid hormones on the control of carbohydrate and lipid metabolism in rat adipose tissue.

Adipose tissue segments excised from normal rats and from rats rendered experimentally hypothyroid and hyperthyroid retained insulin responsiveness when studied in vitro. Basal rates of glucose oxidation to CO2, conversion into glyceride-glycerol, fatty acids, and total lipids, and the activities of pyruvate dehydrogenase and fatty acid synthetase were enhanced in fat pads from hyperthyroid rats when compared with values seen with tissue from euthyroid animals. The response of each of these parameters was further enhanced by treating tissue from hyperthyroid rats in vitro with insulin. Basal rates of glucose oxidation and the activities of pyruvate dehydrogenase and fatty acid synthetase were depressed as a result of hypothyroidism. However, all of these values could be restored to levels approaching the values seen in the basal state for euthyroid rats when tissue segments from hypothyroid rats were incubated in vitro with insulin. The basal rates for glucose conversion into glyceride-glycerol, fatty acids, and total lipids were not changed by hypothyroidism but retained insulin responsiveness. These data suggest that the insulin-effector system in adipose tissue is not altered by thyroid status and that thyroid hormones may act independently with insulin to regulate glucose and lipid metabolism in this tissue at multiple intracellular metabolic sites.

The regulation of adipose tissue pyruvate dehydrogenase activity of dietary fiber.

In vitro studies have established that insulin enhances the oxidation of pyruvate to acetyl CoA by the stimulation of mitochondrial pyruvate dehydrogenase (PDH) activity through plasma membrane binding response (Jarett and Seals 1979; Kiechle, Jarett, Dennis and Kotagal 1980). In the present study adipose tissue PDH activity was utilized as a marker for insulin responsiveness. The metabolic response of this enzyme to exogenous insulin was employed to test the hypothesis that dietary fiber enhances tissue responsiveness to insulin using adipose tissue from male weanling Sprague Dawley rats. Eight groups of rats (n = 5 per group) were fed ad libitum various diets containing different levels of cellulose and protein as already reported elsewhere (Ogunwole, Knight, Adkins, Thomaskutty and Pointer 1985). Percent insulin stimulation of PDH from basal activity (PDS) was utilized as an index of insulin responsiveness. Compared to all fiber treated groups, both basal (PDB) and insulin stimulated (PDI) activities were significantly lower (P less than 0.05) in the fiber free groups at both low (10%) and high (20%) protein levels. At all fiber levels tested (0, 5, 15 and 30%) protein intake resulted in a significant increase in both PDB and PDI. Gradual increase in cellulose intake resulted in a biphasic increase in PDS in both protein groups at the 5% and 30% fiber levels. PDS was higher (P less than 0.05) in the 10% protein groups than the 20% protein group at all fiber levels tested. A significant interaction effect of protein and fiber was observed on PDB (P less than 0.001) and PDI (P less than 0.04) when caloric intake was held constant as a covariate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of fructose, levamisole and vanadate on insulin action in rat adipose tissue.

The effects of dietary fructose, levamisole and vanadate on insulin-stimulated conversion of D-[U-14C]glucose to 14CO2 and to 14C-labeled lipid were examined in rat epididymal adipose tissue. Male weanling rats were fed isoenergetic diets containing either 27% (wt/wt) fructose or glucose for 11 wk. During the final 4 wk, rats in each group were either untreated (control) or treated orally with 20 mg/kg body wt levamisole or 0.5 ppm vanadate via their drinking water. Basal glucose oxidation to CO2 was 45% higher in the fructose-control than in the glucose-control group. Insulin-stimulated glucose oxidation in both control groups was not higher than the basal rate in fructose-fed rats. Basal lipogenesis was 31% lower in the fructose-control than in the glucose-control group. Insulin-stimulated lipogenesis was much higher than basal, but was not different between fructose- and glucose-control groups. Levamisole increased basal lipogenesis in fructose-fed rats. Vanadate acted synergistically with fructose in greatly diminishing insulin-stimulated glucose oxidation. Fasting plasma insulin levels were lower and fasting plasma glucose and triglycerides were higher in fructose- than in glucose-fed rats, irrespective of treatment. Results suggest that adaptation to dietary fructose enhances basal oxidative capacity in an insulin-like fashion and reduces the basal lipogenic capacity of adipose tissue. Treatment with levamisole and vanadate lowers the overall rate of glucose metabolism and alters the effects of fructose.

Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase.

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.

Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation, and respiratory inhibitors.

When isolated rat epididymal fat cells were incubated with [125I]iodoinsulin for 5 min at 37 degrees, radioactivity accumulated in the plasma membrane fraction (Peak 1) and an unidentified particulate fraction (Peak 2) as reported previously (Kono, T., Robinson, F.W., and Sarver, J.A. (1975) J. Biol. Chem. 250, 7826-7835). This accumulation of radioactivity in Peak 2 (but not that in Peak 1) was greatly impaired when cells were incubated with iodoinsulin in the presence of a variety of metabolic inhibitors that reduce the cellular content of ATP. The reduction in the ATP level coincided with a disappearance of the stimulatory effects of insulin on sugar transport and the hormone-sensitive phosphodiesterase. In contrast, ATP depletion had no significant effects, at least during a 5-to 15-min incubation, on the intracellular water space and on the basal sugar transport and phosphodiesterase activities. When cells once depleted on ATP by treatment with 2,4-dinitrophenol (1 mM; 10 min) were washed and suspended in fresh buffer, the ATP level was recovered almost fully in 10 min. This recovery coincided with the restoration of responsiveness to insulin. When cells were incubated with [125I]iodoinsulin or insulin for 5 min at 15 degrees instead of 37 degrees, a negligible quantity of radioactivity accumulated in Peak 2 and insulin failed to activate sugar transport. In contrast, under the same conditions, radioactivity accumulated in Peak 1 and insulin stimulated phosphodiesterase considerably. These results suggest that ATP, or some other compound metabolically related to ATP, may be necessary for the actions of insulin on sugar transport and phosphodiesterase. ATP, or some other related compound, may also be necessary in the formation of the radioactive Peak 2, although the physiological function and cellular location of this peak are yet to be ascertained.

Studies on the role of cyclic guanosine 3':5'-monophosphate and extracellular Ca2+ in the regulation of glycogenolysis in rat liver cells.

Catecholamines increased guanosine 3':5'-monophosphate (cyclic GMP) accumulation by isolated rat liver cells. The increases in cyclic GMP due to 1.5 muM epinephrine, isoproterenol, or phenylephrine were blocked by phenoxybenzamine but not by propranolol. The possibility that cyclic GMP is involved in the glycogenolytic action of catecholamines seems unlikely since cyclic GMP accumulation is also elevated by carbachol, insulin, A23187, and to a lesser extent by glucagon. Furthermore, carbachol had little effect on glycogenolysis while insulin actually inhibited hepatic glycogenolysis. The rise in cyclic GMP due to carbachol was abolished by atropine and that due to all agents was markedly reduced by the omission of extracellular calcium. However, the glycogenolytic action of glucagon and catecholamines was only slightly inhibited by the omission of calcium. The only agent which was unable to stimulate glycogenolysis in calcium-free buffer was the divalent cation ionophore A23187. There was a drop in ATP content of liver cells during incubation in calcium-free buffer which was accompanied by an inhibition of glucagon-activated adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The presence of calcium inhibited the rise in adenylate cyclase activity of lysed rat liver cells due to glucagon or isoproterenol but not that due to fluoride. These results suggest that the stimulation by catecholamines and glucagon of glycogenolysis is not mediated through cyclic GMP nor does it depend on the presence of extracellular calcium. Cyclic GMP accumulation was increased in liver cells by agents which either inhibit, have little affect, or accelerate glycogenolysis. The significance of elevations of cyclic GMP in rat liver cells remains to be established.

Cyclic nucleotides and gluconeogenesis by rat liver cells.

Gluconeogenesis from lactate, pyruvate, fructose, alanine, and other substrates was accelerated by glucagon or epinephrine in hepatocytes isolated from rat liver. Glucagon and epinephrine also increased cyclic AMP accumulation by rat hepatocytes. Isoproterenol increased cyclic AMP but not gluconeogenesis, while phenylephrine accelerated gluconeogenesis. The activation of gluconeogenesis by epinephrine was unaffected by propranolol but blocked by dihydroergotamine. Dibutyryl cyclic AMP added to hepatocytes stimulated gluconeogenesis at concentrations as low as 1 muM. Exogenous cyclic GMP (0.1- muM) inhibited gluconeogenesis due to either glucagon or epinephrine without affecting basal gluconeogenesis. However, carbamylcholine did not affect gluconeogenesis by hepatocytes. Basal gluconeogenesis and the increases due to all agents were inhibited by removal of extracellular calcium or the presence of A-23187, D-600, or tetracaine. In contrast, added 0.1 muM cyclic GMP, 2 mM NH-4-Cl, and 10 muM phenethylbiguanide inhibited glucagon- or epinephrine-stimulated gluconeogenesis without affecting basal values. Studies with hepatocytes indicate that the hormonal activation of gluconeogenesis is not limited to substrates entering prior to triose phosphate formation. Glucagon may act by increasing cyclic AMP which acts via unknown mechanisms to increase gluconeogenesis. In contrast, epinephrine acts via a cyclic AMP-independent mechamism which does not appear to involve cyclic GMP, Ca-2+ flux, of K+ flux.