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A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.

A rapid method for the purification of rat liver phosphorylase kinase 30,000-fold over homogenate values is described. The method allows the isolation of a near homogeneous preparation of phosphorylase kinase initially associated with the glycogen pellet to be accomplished within 24 h. The enzyme has Mr (apparent) = 1.3 million by gel filtration and is composed of subunits similar in size to those of skeletal muscle phosphorylase kinase. The enzyme is phosphorylated by the cAMP-dependent protein kinase: phosphate is incorporated into two of the subunits (Mr = 140,000 and Mr = 116,000) and is closely paralleled by activation of the enzyme. The enzyme is partially inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and is stimulated by 10(-8)-10(-6) M Ca2+. The pH optimum of the nonactivated enzyme is 7.0. Activation by cAMP-dependent protein kinase does not appear to alter the Ca2+ sensitivity of the enzyme. However, it results in a large increase in activity at pH 7 through 8, but not at pH below 6.5. Purified rat liver phosphorylase kinase thus shows many similarities to purified skeletal muscle phosphorylase kinase, but differs in respect to its incomplete inhibition by ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and to the effects of phosphorylation by cAMP-dependent protein kinase on its pH activity profile and Ca2+ sensitivity.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy

The guanylate cyclase reaction was studied to determine the identity of the product(s) formed other than guanosine-3′,5′-monophosphate (cyclic GMP). Partially purified guanylate cyclase preparations from rat lung catalyzed the formation of nearly equal amounts of PP 1 and of cyclic GMP from GTP. Column chromatography of the enzyme preparation on DEAE-Sephadex or Bio-Gel A-5m failed to separate the enzyme(s) involved in formation of cyclic GMP and of PP 1 . Nucleotide inhibitors of cyclic GMP formation also inhibited PP 1 formation, and Ca 2+ , a stimulant of cyclic GMP formation in the presence of Mn 2+ , also stimulated PP 1 formation. Detectable PP 1 formation was not observed when ATP was present instead of GTP. The results show that guanylate cyclase, in vitro , catalyzes the formation of pyrophosphate from GTP concomitant with the synthesis of cyclic GMP.

Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. A great deal of compelling evidence supports the hypothesis that fructose 2,6-bisphosphate plays a key role in the hormonal and substrate regulation of substrate cycling at the fructose 6-phosphate/fructose 1,6-bisphosphate level in liver. This regulation is exerted at the level of the enzyme activities responsible for the synthesis and degradation of fructose 2,6-bisphosphate. Synthesis of the compound is catalyzed by a unique enzyme which transfers the gamma-phosphate of ATP to the C2 position of fructose 6-phosphate (ATP:D fructose 6-phosphate 2-phosphotransferase) while degradation is catalyzed by a phosphohydrolase activity which is specific for the C-2 position of fructose 2,6-bisphosphate (D-fructose 2,6-bisphosphate 2-phosphohydrolase). These activities are distinct from the classical 6-phosphofructo 1-kinase and fructose 1,6-bisphosphatase with regard to molecular weight, interaction with ligands, and the efficiency with which phosphoryl transfer occurs. Both activities have been purified to homogeneity and have been shown to be present in a single enzyme protein, i.e. the enzyme is bifunctional. Incubation of the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase with cAMP-dependent protein kinase and ATP leads to phosphorylation of the enzyme resulting in inactivation of the phosphotransferase activity and stimulation of the phosphohydrolase activity. Since fructose 2,6-bisphosphate is not further metabolized and can only be recycled to fructose 6-phosphate, simultaneous modulation of the synthesis and degradation of the compound by covalent modification of a single protein provides a very efficient and sensitive regulatory mechanism. The bifunctional enzyme was also shown to possess an ATPase activity which was nearly equal to the activity of the kinase reaction. However, in the presence of fructose 6-phosphate the enzyme did not transfer phosphate to water but rather to the C-2 position of the phosphorylated sugar. The ability of the enzyme to catalyze a partial reaction at a rate nearly equal to that of the forward reaction suggested that the reaction mechanism of the kinase proceeds by a two step transfer, i.e. via a phosphoryl enzyme intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.

The inhibition of hepatocyte 6-phosphofructo-1-kinase by glucagon was suppressed by insulin when the enzyme was measured in crude extracts. However, no effect of either hormone was observed after the removal of allosteric effectors from the enzyme, suggesting that the alterations in activity may be due to changes in the level of fructose 2,6-bisphosphate, a potent allosteric activator of the enzyme. Insulin opposed the action of both glucagon and exogenous cyclic AMP to lower fructose 2,6-bisphosphate levels. The concentration of glucagon and of cyclic AMP that gave a half-maximal decrease in fructose 2,6-bisphosphate levels was increased in the presence of 10 nM insulin from 0.03 to 0.09 nM and from 12 to 36 microM, respectively. Insulin also counteracted the effect of maximal concentrations of epinephrine on fructose 2,6-bisphosphate levels. In the presence of 0.02 nM glucagon or 10 microM epinephrine, 10 nM insulin enhanced 6-phosphofructo-2-kinase and decreased fructose 2,6-bisphosphatase activity in (NH4)2SO4-treated hepatocyte extracts. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was shown to be a substrate for the cAMP-dependent protein kinase but not for phosphorylase kinase. It was concluded that insulin opposed the action of glucagon and epinephrine by affecting the phosphorylation state of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Fructose 2,6-bisphosphate levels were decreased in liver cells from diabetic rats. Addition of 30 mM glucose elevated fructose 2,6-bisphosphate levels in cells from fed and 24-h-starved rats but not in cells from diabetic rats. This was probably due to decreases in both 6-phosphofructo-2-kinase and glucokinase activity in the diabetic state. These results show that insulin has both short and long term effects on fructose 2,6-bisphosphate metabolism in liver.

The Chrisman-Snook procedure for instability of the lateral ankle ligaments, first described in 1969, reconstructs the anterior talofibular ligament and the calcaneofibular ligament using one-half of the peroneus brevis tendon, routed through tunnels in the fibula and calcaneus. In the present long-term evaluation of the results of this procedure, forty-eight of sixty ankles, in fifty-seven patients, were assessed after a mean follow-up of ten years (range, four to twenty-four years). The results were excellent in thirty-eight ankles, good in seven, fair in two, and poor in one. The two ankles with a fair result were improved but still had some persistent instability, while the ankle with a poor result (no improvement) was in a patient with generalized ligament laxity. All three patients with a fair or poor result had had a severe reinjury to the ankle. Based on the findings in this study, we concluded that this procedure will restore good long-term function in a high percentage of patients who are disabled by ankle instability due to unhealed or neglected tears of the lateral ligaments.

Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.

A calmodulin-dependent protein kinase purified from liver catalyzed the incorporation of up to 0.7 mol of phosphate per mol subunit of phenylalanine 4-monooxygenase. The phosphorylation was accompanied by a proportional increase in the hydroxylase activity. The reaction was Ca2+-dependent and was inhibited by physiological concentrations of phenylalanine. Phenylalanine 4-monooxygenase was also a substrate for the cGMP-dependent protein kinase, but in this system phenylalanine stimulated the rate of phosphorylation to a similar extent as that observed in the reaction catalyzed by cAMP-dependent protein kinase. The hydroxylase was not a substrate for phosphorylase kinase. The calmodulin-dependent reversal of the kinase reaction in the presence of MgADP, was also inhibited by phenylalanine. Since the kinetics of the reverse reaction was the same using 32P-hydroxylase phosphorylated by calmodulin-dependent and cAMP-dependent kinases, it is likely that both kinases phosphorylate the same site on the enzyme. This conclusion was further supported by peptide mapping of tryptic and peptic digests of 32P-hydroxylase, which revealed one major phosphopeptide with enzyme phosphorylated by either kinase. The Ca2+-dependent and calmodulin-dependent phosphorylation described above may mediate the increased phosphorylation of the hydroxylase [Garrison, J. C., Johnsen, D. E., and Campanile, C. P. (1984) J. Biol. Chem. 259, 3283-3292] and its increased activity [Fisher, M. J., Santana, M. A., and Pogson, C. I. (1984) Biochem. J. 219, 87-90] recently observed in hepatocytes exposed to Ca2+-elevating agents.

A placebo or the antimalarial agent hydroxychloroquine (Plaquenil), which inhibits platelet adhesiveness, and, to a lesser extent, platelet aggregation, was given to 100 patients between forty and ninety-five years old (average age, seventy-four years) who had either fractures or orthopaedic operations involving the skeleton between the knee and the pelvis. Medication was started at the time of admission in a blind, randomized way, fifty patients receiving hydroxychloroquine (200 milligrams three times a day) and fifty, a placebo. No untoward bleeding complications were noted in the treated group. Eight instances of thromboembolism were encountered in the control group as compared with one in the hydroxychloroquine-treated group, a statistically significant difference at the 1.5 per cent level.

Searches for unstable neutral and charged heavy leptons, N and L+-, and for excited states of neutral and charged leptons, nu*, e*, mu*, and tau*, have been performed in e+e- collisions using data collected by the OPAL detector at LEP. The data analysed correspond to an integrated luminosity of about 58pb-1 at a centre-of-mass energy of 183GeV, and about 10pb-1 each at 161GeV and 172GeV. No evidence for new particles was found. Lower limits on the masses of unstable heavy and excited leptons are derived. From the analysis of charged-current, neutral-current, and photonic decays of singly produced excited leptons, upper limits are determined for the ratio of the coupling to the compositeness scale, f/Lambda, for masses up to the kinematic limit. For excited leptons, the limits are established independently of the relative values of the coupling constants f and f'. Searches for unstable neutral and charged heavy leptons, N and L+-, and for excited states of neutral and charged leptons, nu*, e*, mu*, and tau*, have been performed in e+e- collisions using data collected by the OPAL detector at LEP. The data analysed correspond to an integrated luminosity of about 58pb-1 at a centre-of-mass energy of 183GeV, and about 10pb-1 each at 161GeV and 172GeV. No evidence for new particles was found. Lower limits on the masses of unstable heavy and excited leptons are derived. From the analysis of charged-current, neutral-current, and photonic decays of singly produced excited leptons, upper limits are determined for the ratio of the coupling to the compositeness scale, f/Lambda, for masses up to the kinematic limit. For excited leptons, the limits are established independently of the relative values of the coupling constants f and f'.

Publisher Summary The method of using pairs of cAMP analogs to identify cA PK-mediated responses in intact tissues appears to be generally applicable. A number of different cA PK-mediated responses have been demonstrated in several different cell preparations from several different species. These include lipolysis and fatty acid transport in isolated rat adipocytes; phosphorylase activation in rat adipocytes, rat hepatocytes, and bovine neutrophils; phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and mRNA PEPCK induction in continuous cultures of H4 hepatoma cells; and progesterone synthesis and luteinizing hormone (LH) receptor induction in primary cultures of porcine granulose cells. When type I-directed or type II-directed analog pairs are used in vitro, only the appropriate, single isozyme is synergistically activated. This is true for the analog pairs with asterisks when testing a number of isozymes. These include the type I isozyme from rat hepatocytes, bovine neutrophils, rabbit and rat skeletal muscle, and dog thyroid; and the type II isozyme from rat adipocytes, rat hepatocytes, bovine neutrophils, bovine heart, rat skeletal muscle, H4 hepatoma cells, rat liver membranes, and dog thyroid.

Using a computerized pendulum device for delivery of a moderate, reproducible impact on the surface of canine femoral cartilage, the lipids of the cartilage were analyzed by gas chromatography, and a four-fold increase in arachidonic acid was found in the phospholipid pool, as compared with controls. This is the first evidence presented thus far which clarifies the link between mechanical injury and the early biochemical changes of the osteoarthritic process. Electron microscopy of impacted cartilage cells showed an increase in lipids and microtubules and cytoplasmic derangement. There was a brief rise in hexosamine content soon after impaction, followed by a trend toward loss.

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.

The Hall model of experimental arthritis of the knee (excision of the lateral femoral condyle) offers a unique way of investigating the earliest changes of degenerative arthritis. Predictable degeneration of the lateral tibial plateau occurs when there is lack of contact with an opposing surface. Previous work by Hall and Thompson has not concentrated upon the early changes in proven mature animals of optimum age for a study of simulated osteoarthritis. In this work using mature rabbits, two important findings are documented. The earliest observed change in the joint, beginning at 7 days is loss of matrix protein-polysaccharides from the lateral plateau, with no evidence of change in the subchondral bone until much later; in this respect mature differ from immature animals. Furthermore, there is strong evidence for late (three to 6 months) protein-polysaccharide synthesis in the previously degeneraing articular cartilage in which the residual cells reconstitute an effective matrix.

Short-term incubations of intact canine articular cartilage slices with prostaglandins E1 and E2 caused significant losses of hexosamine from cartilage matrix compared to controls. Chloroquine, an inhibitor of DNA primer, prevented this prostaglandin-induced hexosamine depletion. These data suggest that E-prostaglandins may degrade articular cartilage directly through DNA-dependent RNA synthesis of cathepsin-like proteases. Catabolism of articular cartilage probably involves degradation of existing matrix in addition to inhibition of synthesis.

Recently developed high photon yielding fibers that are coupled to position sensitive photomultipliers provide good angular precision and good energy resolution in detecting gamma-rays. The UTD/UCLA High Resolution Gamma Ray Telescope consists of a scintillating plastic fiber Compton or Pair-Production Converter, a time projection gas chamber and scintillating fiber calorimeter. The calorimeter is designed to cover 7c-ray energies from 1 MeV to > 20 GeV. The space-based telescope will be used for detecting i-rays from point-like astrophysical sources and diffused cosmic 1-rays. We present here the description of the telescope, and some preliminary results from a prototype Compton converter.

Seventy-five patients ages 40 to 95 (average, 78), 60 of whom were women and 15 men, were admitted with upper femoral fractures and participated in a study designed to evaluate the possible prophylactic effects of platelet-inhibiting drugs on the incidence of thromboembolism. Aspirin, 600 mg, b.i.d., or a combination of the above doses of aspirin and hydroxychloroquine, 300 mg, b.i.d., were used from the time of admission. The study was prospective, consecutive except for a few exclusions, double-blind and randomized. Standard surgical treatment was given by three orthopedists. Venography by the 11th day was the endpoint, although daily examinations and early impedance plethysmography were also used. Twenty-five patients received placebos, 24 aspirin alone and 26 aspirin and Plaquenil. In the control group, 15 of 25 (60%) developed thrombosis and two had nonfatal pulmonary emboli. With aspirin, six of 24 (25%) had thrombosis and one a fatal embolus. In the combination group seven of 26 (27%) developed thrombosis and there were no emboli. No significant difference existed between treatment groups, but there was significant difference between the control and treatment group at the 2% level by the Yates chi2 test. Treated women received significant protection against thromboses (p less than 0.02); 13 of 20 untreated women had deep thromboses versus 12 of 40 in the treated women. Aspirin prophylaxis is significantly useful in fractured hip patients. The combination of aspirin and Plaquenil offers no additional benefit and only leads to complications from hematomas.

The inhibition of hepatocyte 6-phosphofructo-1-kinase by glucagon was suppressed by insulin when the enzyme was measured in crude extracts. However, no effect of either hormone was observed after the removal of allosteric effectors from the enzyme, suggesting that the alterations in activity may be due to changes in the level of fructose 2,6-bisphosphate, a potent allosteric activator of the enzyme. Insulin opposed the action of both glucagon and exogenous cyclic AMP to lower fructose 2,6-bisphosphate levels. The concentration of glucagon and of cyclic AMP that gave a half-maximal decrease in fructose 2,6-bisphosphate levels was increased in the presence of 10 nM insulin from 0.03 to 0.09 nM and from 12 to 36 microM, respectively. Insulin also counteracted the effect of maximal concentrations of epinephrine on fructose 2,6-bisphosphate levels. In the presence of 0.02 nM glucagon or 10 microM epinephrine, 10 nM insulin enhanced 6-phosphofructo-2-kinase and decreased fructose 2,6-bisphosphatase activity in (NH4)2SO4-treated hepatocyte extracts. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was shown to be a substrate for the cAMP-dependent protein kinase but not for phosphorylase kinase. It was concluded that insulin opposed the action of glucagon and epinephrine by affecting the phosphorylation state of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Fructose 2,6-bisphosphate levels were decreased in liver cells from diabetic rats. Addition of 30 mM glucose elevated fructose 2,6-bisphosphate levels in cells from fed and 24-h-starved rats but not in cells from diabetic rats. This was probably due to decreases in both 6-phosphofructo-2-kinase and glucokinase activity in the diabetic state. These results show that insulin has both short and long term effects on fructose 2,6-bisphosphate metabolism in liver.

The Chrisman-Snook procedure for instability of the lateral ankle ligaments, first described in 1969, reconstructs the anterior talofibular ligament and the calcaneofibular ligament using one-half of the peroneus brevis tendon, routed through tunnels in the fibula and calcaneus. In the present long-term evaluation of the results of this procedure, forty-eight of sixty ankles, in fifty-seven patients, were assessed after a mean follow-up of ten years (range, four to twenty-four years). The results were excellent in thirty-eight ankles, good in seven, fair in two, and poor in one. The two ankles with a fair result were improved but still had some persistent instability, while the ankle with a poor result (no improvement) was in a patient with generalized ligament laxity. All three patients with a fair or poor result had had a severe reinjury to the ankle. Based on the findings in this study, we concluded that this procedure will restore good long-term function in a high percentage of patients who are disabled by ankle instability due to unhealed or neglected tears of the lateral ligaments.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy

A placebo or the antimalarial agent hydroxychloroquine (Plaquenil), which inhibits platelet adhesiveness, and, to a lesser extent, platelet aggregation, was given to 100 patients between forty and ninety-five years old (average age, seventy-four years) who had either fractures or orthopaedic operations involving the skeleton between the knee and the pelvis. Medication was started at the time of admission in a blind, randomized way, fifty patients receiving hydroxychloroquine (200 milligrams three times a day) and fifty, a placebo. No untoward bleeding complications were noted in the treated group. Eight instances of thromboembolism were encountered in the control group as compared with one in the hydroxychloroquine-treated group, a statistically significant difference at the 1.5 per cent level.

This article describes a conservative program of treatment for patellofemoral pain of chondromalacic origin. It has become obvious that patellofemoral pain syndromes are derived from cartilage degradation and from soft-tissue changes, including tendinitis and neuromatous degeneration.

The major problem in osteoarthritis research today is our inability to promote effective cartilage regeneration in the presence of adult chondromalacia. Yet such regeneration is consistently present in acromegalics. The present study of experimentally damaged rabbit knee cartilage measures numbers of cells, mitoses, tritiated thymidine incorporation, DNA content, collagen presence under polarized light and sulfated protein polysaccharides histologically. Under growth hormone stimulation cell counts nearly double as compared to controls, TTI increases, DNA content triples or more. Matrix healing is evident histologically. The addition of salicylate has a mild additive effect on cell counts and on retention of matrix. The above data appear to be the first reported demonstration of these effects of growth hormone on experimentally damaged cartilage and may gain importance for clinical use when human growth hormone or substitutes for it become available.

The mechanism of actions of glucagon, alpha- and beta-adrenergic agonists, vasopressin and angiotensin II in the liver proposed in this article are summarized in Fig. 8. The actions of glucagon and beta-adrenergic agonists in liver can be entirely ascribed to their interaction with specific plasma membrane receptors which activate adenylate cyclase leading to the intracellular accumulation of cAMP and activation of cAMP-dependent protein kinase. This enzyme phosphorylates phosphorylase b kinase, glycogen synthase, L-type pyruvate kinase, and other liver proteins resulting in alterations in their activities which can account for several of the known hepatic responses to glucagon. There is no clear evidence that Ca2+ ions are involved in the hepatic actions of this hormone. Glucocorticoids, but not thyroid hormones, are required for normal responsiveness of the liver to glucagon. The steroids do not modify cAMP accumulation or cAMP-dependent protein kinase activation, but may act by modulating the action of the kinase on its substrates. Glucocorticoids and thyroid hormones decrease beta-adrenergic responses in the liver apparently by decreasing the number of beta-receptors. Insulin inhibits the actions of physiological concentrations of glucagon by decreasing cAMP accumulation: its mechanism of action is unknown. The actions of alpha-adrenergic agonists, vasopressin and angiotensin II on the liver resemble those of glucagon, but do not involve accumulation of cAMP or activation of cAMP-dependent protein kinase. These agents appear to act by increasing cytosolic Ca2+ thus altering the activities of Ca2+-sensitive enzymes such as phosphorylase b kinase and calmodulin-dependent glycogen synthase kinase. Their receptors appear to be located exclusively on the plasma membrane and a major mechanism by which they raise cytosolic Ca2+ is by inducing the release of this cation from mitochondria. These considerations imply the existence of an intracellular messenger(s) for these agents which is generated at the plasma membrane in response to receptor activation and exerts effects on mitochondria or perhaps other intracellular structures. Glucocorticoids and thyroid hormones increase alpha-adrenergic responses in the liver apparently by increasing the number of alpha-receptors. Insulin inhibits the responses of the liver to alpha-agonists, but not to vasopressin or angiotensin II.

A rapid method for the purification of rat liver phosphorylase kinase 30,000-fold over homogenate values is described. The method allows the isolation of a near homogeneous preparation of phosphorylase kinase initially associated with the glycogen pellet to be accomplished within 24 h. The enzyme has Mr (apparent) = 1.3 million by gel filtration and is composed of subunits similar in size to those of skeletal muscle phosphorylase kinase. The enzyme is phosphorylated by the cAMP-dependent protein kinase: phosphate is incorporated into two of the subunits (Mr = 140,000 and Mr = 116,000) and is closely paralleled by activation of the enzyme. The enzyme is partially inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and is stimulated by 10(-8)-10(-6) M Ca2+. The pH optimum of the nonactivated enzyme is 7.0. Activation by cAMP-dependent protein kinase does not appear to alter the Ca2+ sensitivity of the enzyme. However, it results in a large increase in activity at pH 7 through 8, but not at pH below 6.5. Purified rat liver phosphorylase kinase thus shows many similarities to purified skeletal muscle phosphorylase kinase, but differs in respect to its incomplete inhibition by ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and to the effects of phosphorylation by cAMP-dependent protein kinase on its pH activity profile and Ca2+ sensitivity.

Incubation of rat liver phosphorylase kinase in the presence of MgATP results in a time-dependent increase in activity, i.e., activation. Determination of the magnitude of activation depends, in large part, on the relative concentrations of Mg2+ and ATP used in the phosphorylase kinase activity assay, such that as the Mg2+ to ATP ratio increases less activation is detectable. Prior to activation, maximal activity of nonactivated phosphorylase kinase requires a 2–3 fold molar excess of Mg2+ (i.e., free Mg2+) over ATP. MgATP-dependent activation of the enzyme results in an alteration in the free Mg2+ requirement such that the activity of the activated enzyme is sharply inhibited by the free cation. Inhibition by free Mg2+ of the activated enzyme is rapidly reversed by removal of free Mg2+ but is not affected by addition of Ca2+. Both nonactivated and activated forms of enzyme appear to be inhibited by free ATP4–. The results show that the use of high concentrations of free Mg2+ in the phosphorylase kinase activity assay can blunt or completely obscure changes in enzyme activity following activation of the enzyme.

Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.

In a prospective study extending from 1966 to 1974, the results of the Hauser procedure were compared with the Roux-Goldthwait procedure for the correction of recurrent dislocations of the patella. In 87 knees in 75 patients, repaired consecutively and in a random manner with one procedure or the other, 100% retrieval was achieved. The average follow-up period was 7.7 years. Significant complications, though not necessarily of long term, occurred in 24 of 47 knees receiving Hauser repairs as compared with 6 of 40 knees in the Roux-Goldthwait group. Satisfactory long-term results were obtained in 72% with the Hauser technique and 93% with the Roux-Goldthwait technique.

In considering the critical problem of cash flow within the petroleum industry, it is tempting to recite the background and recent trends in the industry, to draw conclusions and then suggest action plans which, if not implemented, would leave the industry and this country in an untenable position. I have resisted this temptation, for the most part, for several reasons. To discuss the problem in that amount of detail with such a group as this would appear to me to be playing an old record, even though that record is important enough to replay as often as possible. But since this is an industry group, it seemed that we would just end up crying on each other's shoulder without really moving ahead on the job of solving the problem. There are two things on which we might usefully spend some time today. The first is further consideration of the one element that could stop the slide of the industry in every respect and set the framework for rebuilding to the point where the industry was 15 years ago. That element is price. Now, price increases have certainly been covered over and over again by industry spokesmen, but I hope to look at the subject of prices from a perspective slightly different from some of the perspective slightly different from some of the things we have recently been hearing and reading. Since whatever the outcome of price movements may be and let us assume it will be a change that would favor the industry's economics that outcome will take some time to come and will take longer to create the climate that we all hope to achieve. This will lead me to the second subject we can examine today, and that is how the financial community might help bridge the interim period. In discussing the first, we need to review briefly the substantive points that summarize the industry. There is not a chief executive or financial officer of a major or medium-sized oil company today who has not at one time or another, formally or informally, expressed his views on the position of the industry and hoped to hit an audience who could either help formulate opinion or take action to help solve the problem. These presentations have been in the form problem. These presentations have been in the form of speeches, paid advertisements, prepared booklets or letters, and position papers directed to the government. The issues are the adequacy of domestic supplies of crude petroleum and natural gas and the adequacy of a supply of capital to develop those resources and bring them to the market. In the area of adequate future domestic supply, you are all familiar with the following facts:Exploratory wells drilled since 1956 have declined steadily.Why? - Adverse economics arising from controlled gas prices, federal jawboning on crude oil and products prices (lately de-facto controls), and substantial cost increases from inflation and rising tax burdens both at home and abroad.This has resulted in declining reserve-to-production ratios. Crude oil has dropped from 21 years' supply in 1960 to 12 years' supply in 1971; and natural gas has dropped from 13 years' to 9 years' over the same span.Projections to 1985 show an inability of other energy sources to make up in time the shortfall in our lagging oil and gas supplies, though it is conceded that the reserves are there at some cost and price.Projections to 1985 under present conditions indicate we well be an importer of about 50 percent of our crude oil requirements, a heavy importer of gas and a user of new and expensive synthetic gas. In the area of adequate future capital to support the industry to 1985, these are the facts:Essentially lower rate of return on invested capital in the decade of the 60's as compared with that available in the 50's falling to 9.3 percent domestically in 1971;Capital spending increasing at faster rates than the growth of internally generated cash;Increasing debt equity ratios from 16 percent of total capital in 1961 to more than 30 percent in 1971 (and nearer to 40 percent if debt equivalents are counted);Taxes increasing at rates much faster than net income; andCeilings on crude and product prices, first by competitive industry pressures and later by governmental decree or political pressure. Most analysts of the problem and spokesmen for the industry have reached the same conclusions that the answers in an important part will come from the role of the government and the impact of its future actions. P. 53

Neck injuries are infrequent in college athletes compared to extremity injuries but are especially important because of their serious potential. Schneider 1 has discussed the possibility of fatal outcome from cervical hyperextension in football, when the face bar of the helmet is used as a lever to force the head backward. In our experience, a more common injury is a lateral neck sprain, the so-called nerve pinch in trainers' terminology. The typical injury is a blow on the side of the head, with immediate pain from the base of the neck to the hand, prickly paresthesia of the whole arm, and inability to move the extremity. Within a few minutes, this set of sensations disappears, leaving a dull ache in the neck and shoulder of the affected side. Within a few more minutes, the injured player is seeking and often obtains permission to return to the game. Only later does