A simple drawing test to identify patients who are unlikely to be able to learn to use an inhaler.

Previous research has shown that patients are not able to learn to use self-administered inhaler devices if they have impaired general cognition, executive (frontal lobe) dysfunction or dyspraxia. These impairments are most frequently encountered in frail elderly people. Some of the tests required to detect such impairments are time-consuming and are therefore not easily adopted in routine clinical practice, particularly in an outpatient setting. We performed a study of 50 elderly inhaler-naïve patients to explore the use of a simple quick drawing test (copying overlapping pentagons) to determine whether that test was able to identify patients who were unable to learn to use a Turbohaler. Patients who were not able to perform the pentagon copying test were found to be unable to learn to use a Turbohaler with a specificity of 93% and positive predictive value of 87%. This was at least as discriminating as the Abbreviated Mental Test and the Mini Mental State Examination. The overlapping pentagon drawing test is useful in clinical practice to identify patients who will probably not use a self-administered inhaler correctly, and who will require an alternative approach to the treatment of their airways disease. This finding might have implications for other self-administered treatment, such as insulin pens and complex drug regimens.

BRL37344, but not CGP12177, stimulates fuel oxidation by soleus muscle in vitro.

The beta(3)-adrenoceptor agonist, (RR+SS)-(+/-)-4-[2-)2-)3-chlorophenyl)-2-hydroxyethyl)amino)propyl]ph enoxyacetate (BRL37344), stimulated fuel utilisation by isolated mouse soleus muscle at concentrations 10- to 100-fold lower than those required to stimulate lipolysis in brown adipocytes. At 1x10(-10) M BRL37344, uptake and phosphorylation of 2-deoxyglucose was increased (40%), as was glucose-oxidation (50%), palmitate-oxidation (70%) and oxidation of [2-14C]pyruvate (2-fold), indicating stimulation of tricarboxylic acid cycle reactions. Oxidation of [1-14C]pyruvate was unaffected, indicating no stimulation of pyruvate dehydrogenase activity. Other beta(3)-adrenoceptor agonists, disodium(RR)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]- 1,3-benzodioxazole-2,2-dicarboxylate (CL316,243, 1x10(-7) M) and (S)-4-¿2-[2-hydroxy-3-(4-hydroxyphenoxy)propylamino]ethyl¿pheno xymeth ylcyclohexylphosphiric acid lithium salt (SB226552, 1x10(-9) M), achieved similar stimulation of 2-deoxyglucose uptake and phosphorylation but (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP12177A) had no effect. The inhibitor of protein kinase A, H-89 (isoquinolinesulfonamide), had little effect on the stimulation of pyruvate-oxidation by BRL37344, while the specific inhibitor of protein kinase C, bisindolylmaleimide IX, reduced the stimulated rate to slightly below basal values. We consider that these responses provide evidence of the presence of a novel beta-adrenoceptor in skeletal muscle, which we have termed beta(skel)-adrenoceptor.

N-Acetyl-beta-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1.

Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 microM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 microM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 microM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented.

Hydroxycitrate causes altered pyruvate metabolism by tumorigenic cells.

Metabolic fates of pyruvate (CO2, lactate, citrate) in normal and neoplastic cells have been assessed. Pyruvate consumption by tumour cells falls (by 72-85%) and mean percentage oxidation rises from 75% to 91% with hydroxycitrate. Ratios of rates of oxidation of (3-(14)C-pyruvate) : (1-(14)C-pyruvate), indicating CO2 produced from TCA cycle activity : that from PDH activity, are higher for tumorigenic (0.17-0.24) than for non-tumorigenic (0.005-0.04) cells and increase (0.27-0.65 and 0.13-0.29, respectively) with hydroxycitrate. Although maximal ATP-citrate lyase activities do not correlate with malignancy, citrate may be a major fate of glutaminolytic pyruvate in tumour cells. Citrate accounts for 14-37% of consumed glutamine compared with 11-13% being recovered as CO2. By contrast, approximately 100% of glycolytic pyruvate is converted to lactate.

Effects of C-1-substituted glucose analogue on the activation states of glycogen synthase and glycogen phosphorylase in rat hepatocytes.

A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8 mM 1-GlcNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced the dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold improvement on glucose for the liver activity and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP inactivation but failed to elicit activation of GS. Glucose-induced activation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosine analogue and kinase inhibitor, 5'-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylation of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cells at 20 mM 1-GlcNAc but was reduced by the presence of 50 microM ITU (0.775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure of 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.

High Km glucose-phosphorylating (glucokinase) activities in a range of tumor cell lines and inhibition of rates of tumor growth by the specific enzyme inhibitor mannoheptulose.

Differences in modes of control of glycolysis in tumor cells, compared with normal cells, have suggested that phosphofructokinase may not catalyse the rate-controlling step. Instead, hexokinase activity may assume a more important regulatory role. Hexokinase activities are consistently lower than those of phosphofructokinase in tumor cells, and the former enzyme may be saturated with its substrate (M. Board et al., Biochem. J. 265: 503-509, 1990). The present work has focused on the glucose-phosphorylation step in tumor cell glycolysis. A range of eight human tumor cell-lines, one human tumor tissue, and four rat tumor cell lines were found to have an additional glucose-phosphorylating activity, with properties similar to hepatic glucokinase. Maximal activities range from 1.1-20 nmol/min/mg cell protein, and the activity is consistently absent from any untransformed cell line or tissue tested, except rat liver tissue (18 nmol/min/mg cell protein). Tumor cell glucokinase activity has been characterized by its high Km for glucose (8-11.8 mM); inhibition by the specific glucokinase inhibitor, mannoheptulose (I50, 12.5 mM); and lack of inhibition by 10 mM glucose-6-phosphate. Mannoheptulose also causes inhibition of glucose uptake by tumor cells (25-75% at 30 mM mannoheptulose) and inhibition of rates of growth of cultured tumor cell lines (I50, 21.4 mM). Rates of growth of human tumors in experimental animals are dramatically reduced (by 65-79%) by a dose of 1.7 mg/g mannoheptulose daily for 5 days. The potential of the naturally occurring sugar, mannoheptulose (which is purified from avocados and is assumed to be of low toxicity), as a cancer treatment is discussed.

Comparison of disposable and glass mercury thermometers.

The aim of this study was to compare the accuracy of disposable thermometer recordings with those from a mercury thermometer. The disposable thermometer has heat-sensitive clinical dots that change colour to indicate temperature measurement. The trial provided fascinating information, questioning current nursing practice and the safety of the tools we use. We need to challenge when and with what we record patients' temperatures. The results from the trial suggested that not only did the disposable thermometer record quicker than the mercury thermometer but that it was as accurate and cheaper.

Effects of novel analogues of D-glucose on glycogen phosphorylase activities in crude extracts of liver and skeletal muscle.

The inhibitory properties of a series of both N-linked and C-linked C1-substituted glucose derivatives towards glycogen phosphorylase (GP) activity from crude extracts of rat liver and muscle have been measured. The most effective inhibitor was N-acetyl-beta-D-glucopyranosylamine, which has Kis of 51 microM (muscle GPa), 30 microM (muscle GPb), 2.7 mM (liver GPa) and 4 mM (liver GPb). All analogues tested inhibit muscle GP more potently than liver GP, highlighting some differences between the two isoenzymes, which are nearly 80% similar. The human liver GP enzyme has been modelled on the basis of the rabbit muscle structure and, together with comparison of structures of muscle GPa and GPb, has provided some insights into possible explanations for the different properties of the two isoenzymes. Maximal activities of GP have also been measured in tissues from diabetic (db/db) and wild-type (db/+) mice. Liver GP from db/db mice exhibits higher activity [132% (a)-67% (b)] than from db/+ controls, although similar activities were observed for muscle GP from both db/db and db/+ animals.

Application of metabolic-control logic to fuel utilization and its significance in tumor cells.

The importance and the value of applying metabolic-control logic to the question of fuels, their rates of utilization and their significance to the process of proliferation are presented. Application of the recently developed quantitative theory of metabolic control of branched pathways provides a hypothesis to account for the high rate of both glycolysis and glutaminolysis in lymphocytes, macrophages and, in particular, in tumor cells. Both glycolysis and glutaminolysis provide metabolic intermediates for biosynthetic pathways: for example, glucose-6-phosphate for the formation of ribose-5-phosphate, and glutamine, ammonia and aspartate which are required for the synthesis of purine and pyrimidine nucleotides. However, the rates of both glycolysis and glutaminolysis are greatly in excess (greater than 400-fold) of the requirements for the biosynthetic processes. If energy formation per se was the major reason for the high rate of glutamine utilization, why is the oxidation only partial? The ability of the cell to divide will require the synthesis of all the DNA, RNA, phospholipids, etc., at precise times in the cell cycle. Hence very high and accurate sensitivity of the processes that provide the precursors for these compounds to their specific regulators will be expected. Maintenance of high rates of glycolysis and glutaminolysis at all times can be seen therefore as a device to allow intermediates to be "tapped off" at the precise rate required whenever they are needed for biosynthesis. Maximal activities of some key enzymes of glycolysis, the tricarboxylic acid cycle and glutaminolysis from a variety of normal, neoplastic and suppressed cells are presented. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in nontumorigenic cells and higher in metastatic cells than in tumorigenic cells: for nontumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells.

1. Maximal activities of some key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and glutaminolysis were measured in homogenates from a variety of normal, neoplastic and suppressed cells. 2. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. 3. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in non-tumorigenic cells and higher in metastatic cells than in tumorigenic cells: for non-tumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. 4. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8. 5. Both citrate synthase and isocitrate dehydrogenase activities are present in non-neoplastic and neoplastic cells, suggesting that the full complement of tricarboxylic-acid-cycle enzymes are present in these latter cells. 6. In neoplastic cells, the activity of glutaminase is similar to or greater than that of hexokinase, which suggests that glutamine may be as important as glucose for energy generation in these cells.