Decreased myocardial adenyl cyclase activity in hypothyroidism

It has been suggested that hypothyroidism may alter the responsiveness of the heart to sympathetic stimulation. To define more precisely the interrelationship between hypothyroidism and catecholamine responsiveness we: (a) studied the effects of norepinephrine and fluoride on the activation of adenyl cyclase in the particulate fraction of heart homogenates from euthyroid and hypothyroid cats; and (b) assessed the contractile response of isolated right ventricular papillary muscles from the same cats to increasing concentrations of norepinephrine. It was found that maximal accumulation of cyclic 3',5'-adenosine monophosphate (3',5'-AMP) was significantly lower at peak norepinephrine concentrations in the hypothyroid (284 +/-5 pmoles) than in the euthyroid group (326 +/-10 pmoles) (P < 0.02). However, the K(m) for norepinephrine was similar in both groups (1-2 x 10(-5) moles/liter), and there was no apparent change in the threshold concentration. Fluoride-mediated increases in Cyclic 3',5'-AMP accumulation were also significantly lower in the hypothyroid (585 +/-25 pmoles) as compared to the euthyroid group (790 +/-20 pmoles) (P < 0.02). In contrast, norepinephrine produced a similar augmentation of contractility in isolated papillary muscles from the hypothyroid and euthyroid cats. It thus appears that although the hypothyroid state is associated with a decrease in the total amount of myocardial adenyl cyclase per milligram of tissue capable of being activated by norepinephrine or fluoride, there is no change in the sensitivity of the enzyme to norepinephrine stimulation. Moreover, the finding that the inotropic response to norepinephrine is unaltered in hypothyroidism is compatible with the hypothesis that only a fraction of the total intracellular cyclic 3',5'-AMP produced by norepinephrine activation of adenyl cyclase is required to elicit the inotropic response.     

Alkaline Phosphatase. POSSIBLE INDUCTION BY CYCLIC AMP AFTER CHOLERA ENTEROTOXIN ADMINISTRATION

The present studies were undertaken to determine the role, if any, of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) as a chemical inducer of rat liver alkaline phosphatase. Cholera enterotoxin, given intravenously to rats, led to a rapid rise in the activity of hepatic adenyl cyclase that was 7½ times greater than control values in 6 h. Cyclic AMP levels were also significantly increased above control values while the activity of cyclic nucleotide phosphodiesterase was unchanged. Hepatic alkaline phosphatase activity was increased 5½ times above control in 12 h, but its rise followed that of adenyl cyclase and cyclic AMP by several hours. Cycloheximide inhibited the rise of hepatic alkaline phosphatase but not that of adenyl cyclase. The administration of glucagon, a known stimulator of hepatic adenyl cyclase, and of dibutyryl cyclic AMP, led to similar striking increases in hepatic alkaline phosphatase activity. This alkaline phosphatase increase was blocked by the prior administration of cycloheximide. Bile duct ligation, a known stimulator of hepatic alkaline phosphatase activity, failed to produce any significant changes in adenyl cyclase or cyclic AMP. Concomitant treatment of rats with bile duct ligation and cholera enterotoxin or bile duct ligation and glucagon, had no additive effect on the increase in hepatic alkaline phosphatase activity, although the increase occurred earlier. These results suggest that: (a) cyclic AMP may act as an inducer of hepatic alkaline phosphatase: (b) the stimulation of hepatic alkaline phosphatase by cholera enterotoxin is mediated by cyclic AMP; (c) the rise in hepatic alkaline phosphatase following bile duct ligation is not mediated by cyclic AMP; (d) the same alkaline phosphatase in rat liver may be induced by two (or more) mechanisms, only one of which requires cyclic AMP.     

Activation of myocardial adenyl cyclase by histamine in guinea pig, cat, and human heart

Histamine has positive inotropic and chronotropic effects on the heart which are not abolished by beta adrenergic-blocking agents. Since the positive inotropic and chronotropic effects of other hormones on the heart are thought to be mediated by cyclic 3',5'-AMP, we examined the effect of histamine on adenyl cyclase in particulate preparations of guinea pig, cat, and human myocardium. Histamine at the peak of its dose-response curve, 3 x 10(-4)moles/liter, produced approximately a 300% increase in cyclic 3',5'-AMP accumulation in the guinea pig, 60% in the cat, and 90% in the human heart particles. Half-maximal activity for the histamine mediated activation of adenyl cyclase in the guinea pig was 9 x 10(-6)moles/liter, almost identical with that observed for norepinephrine in the same preparation. DL-Propranolol, 1 x 10(-5)moles/liter, did not abolish the activation of adenyl cyclase produced by histamine but did abolish the activation produced by norepinephrine. In contrast, diphenhydramine hydrochloride, Benadryl, 8 x 10(-5)moles/liter, abolished the activation of adenyl cyclase by histamine but not that produced by norepinephrine. These data suggest that there are at least two receptor sites in guinea pig heart mediating the activation of adenyl cyclase, one responsive to histamine, the other to norepinephrine. In addition, combined maximal doses of histamine and norepinephrine produced completely additive effects on the activation of adenyl cyclase, which suggests that at least two separate adenyl cyclase systems are present in the heart, each responsive to one of these hormones. However, definitive proof would require physical separation of the two enzymes.     

Influence of Calcium on the Inotropic Actions of Hyperosmotic Agents, Norepinephrine, Paired Electrical Stimulation, and Treppe

To analyze the interaction of calcium ion concentration with hypertonic agents and with other inotropic interventions, isolated right ventricular cat papillary muscles were studied under isometric conditions in Krebs-Ringer bicarbonate solution. Extracellular calcium concentrations were varied between 2.5 and 11.0 mM. Maximal inotropic effects occurred between 5 and 8.0 mM calcium and further elevation to 11.0 mM was without additional influence. The effect of hyperosmotic sucrose and mannitol on papillary muscle performance was compared with that of 10(-6) M norepinephrine at calcium concentrations of 2.5 and 10.0 mM and with paired electrical stimulation in 10.0 mM calcium. Both norepinephrine and the hyperosmotic agents produced significant increases in developed tension and in the maximal rate of tension rise (dT/dt) in Krebs-Ringer in 2.5 and 4.0 mM calcium. In 10 mM calcium norepinephrine increased developed tension and dT/dt, but sucrose and mannitol caused no change or small reductions in both. Paired electrical stimulation, like hyperosmolality, caused no increase in dT/dt in 10 mM calcium.The presence of a potent pharmacological inhibitor of systolic calcium transfer across the cell membrane (D600, 10(-6) M) reduced developed tension and dT/dt by 76+/-2.7 and 74+/-2.0%, respectively, and prevented and in fact reversed the expected increase in dT/dt associated with an increase in rate of stimulation (treppe). However, hypertonic mannitol and paired pacing persisted in causing marked increases in developed tension and dT/dt even in the presence of D600, suggesting that their inotropic effects are not dependent on increased intracellular transfer of calcium during systole through cell membrane channels in which D600 acts as a competitive inhibitor.The results of these studies suggest that apparent functional saturation of intracellular calcium receptor sites eliminates any additional inotropic effect of hyperosmolality or paired pacing. The data are compatible with the hypothesis that the inotropic effects of hyperosmolality and of paired pacing result from an increase in calcium concentration at the myofilaments during contraction. The increase induced by hyperosmolality might occur because of an increase in the total amount of calcium released into the cytosol with each action potential and/or as a passive consequence of cellular dehydration. Norepinephrine has the capacity to increase contractility even when intracellular calcium receptor sites appear to be functionally saturated, suggesting that it may act at least in part by a mechanism that is independent of changes in net intracellular calcium concentration.     

Myocardial adenyl cyclase: activation by thyroid hormones and evidence for two adenyl cyclase systems

The mechanism responsible for the hyperdynamic circulatory state in hyperthyroidism has not been defined. Although certain cardiac manifestations resemble those caused by excessive adrenergic stimulation, recent evidence suggests that thyroid hormone exerts an effect on the heart that is independent of the adrenergic system. Since the inotropic and chronotropic effects of norepinephrine appear to be mediated by activation of adenyl cyclase, the possibility that thyroxine and triiodothyronine are also capable of activating adenyl cyclase was examined in the particulate fraction of cat heart homogenates.L-thyroxine and L-triiodothyronine increased the conversion of adenosine triphosphate-(32)P (ATP-(32)P) to cyclic 3',5'-adenosine monophosphate-(32)P (3',5'-AMP-(32)P) by 60 and 45% respectively (P < 0.01). A variety of compounds structurally related to the thyroid hormones, but devoid of thyromimetic activity did not activate adenyl cyclase: these included 3,5-diiodo-L-thyronine, L-thyronine, 3,5-diiodotyrosine, monoiodotyrosine, and tyrosine. D-thyroxine activated adenyl cyclase and half maximal activity was identical to that of the L-isomer. Although the beta adrenergic blocking agent propranolol abolished norepinephrine-induced activation of adenyl cyclase, it failed to alter activation caused by thyroxine. When maximal concentrations of L-thyroxine (5 x 10(-6) moles/liter) and norepinephrine (5 x 10(-5) moles/liter) were incubated together, an additive effect on cyclic 3',5'-AMP production resulted.THIS INVESTIGATION DEMONSTRATES: (a) thyroid hormone is capable of activating myocardial adenyl cyclase in vitro and (b) this effect is not mediated by the beta adrenergic receptor. Moreover, the additive effects of norepinephrine and thyroxine suggest that at least two separate adenyl cyclase systems are present in the heart, one responsive to norepinephrine, the other to thyroid hormone.These findings are compatible with the hypothesis that the cardiac manifestations of the hyperthyroid state may, in part, be caused by the direct activation of myocardial adenyl cyclase by thyroid hormone.     

Neuroendocrine responses to glucose ingestion in man. Specificity, temporal relationships, and quantitative aspects

The mechanisms of postprandial glucose counterregulation—those that blunt late decrements in plasma glucose, prevent hypoglycemia, and restore euglycemia—have not been fully defined. To begin to clarify these mechanisms, we measured neuroendocrine and metabolic responses to the ingestion of glucose (75 g), xylose (62.5 g), mannitol (20 g), and water in ten normal human subjects to determine for each response the magnitude, temporal relationships, and specificity for glucose ingestion. Measurements were made at 10-min intervals over 5 h. By multivariate analysis of variance, the plasma glucose (P < 0.0001), insulin (P < 0.0001), glucagon (P < 0.03), epinephrine (P < 0.0004), and growth hormone (P < 0.01) curves, as well as the blood lactate (P < 0.0001), glycerol (P < 0.001), and β-hydroxybutyrate (P < 0.0001) curves following glucose ingestion differed significantly from those following water ingestion. However, the growth hormone curves did not differ after correction for differences at base line. In contrast, the plasma norepinephrine (P < 0.31) and cortisol (P < 0.24) curves were similar after ingestion of all four test solutions, although early and sustained increments in norepinephrine occurred after all four test solutions. Thus, among the potentially important glucose regulatory factors, only transient increments in insulin, transient decrements in glucagon, and late increments in epinephrine are specific for glucose ingestion. They do not follow ingestion of water, xylose, or mannitol.

Following glucose ingestion, plasma glucose rose to peak levels of 156±6 mg/dl at 46±4 min, returned to base line at 177±4 min, reached nadirs of 63±3 mg/dl at 232±12 min, and rose to levels comparable to base line at 305 min, which was the final sampling point. Plasma insulin rose to peak levels of 150±17 μU/ml (P < 0.001) at 67±8 min. At the time glucose returned to base line, insulin levels (49±12 μU/ml) remained fourfold higher than base line (P < 0.01); thereafter they declined but never fell below base line. Plasma glucagon decreased from 95±14 pg/ml to nadirs of 67±11 pg/ml (P < 0.001) at 84±9 min and then rose progressively to peak levels of 114±17 pg/ml (P < 0.001 vs. nadirs) at 265±12 min. Plasma epinephrine, which was 18±4 pg/ml at base line, did not change initially and then rose to peak levels of 119±20 pg/ml (P < 0.001) at 271±13 min.

These data indicate that the glucose counterregulatory process late after glucose ingestion is not solely due to the dissipation of insulin and that sympathetic neural norepinephrine, growth hormone, and cortisol do not play critical roles. They are consistent with, but do not establish, physiologic roles for the counterregulatory hormones—glucagon, epinephrine, or both—in that process.

     

Influence of a phosphodiesterase inhibitor on the chronotropic effects of glucagon and norepinephrine in fetal mouse hearts

Fetal mouse hearts develop tachycardia in response both to norepinephrine and to glucagon, but although adenylate cyclase is stimulated and adenosine 3':5'-monophosphate (cyclic AMP) elevated by norepinephrine, no measurable changes are produced by glucagon. To test further the possible independence of glucagon chronotropy from the cyclic AMP system, the effects of a phosphodiesterase inhibitor were evaluated. The dose-response curve to norepinephrine was shifted to the left by the phosphodiesterase inhibitor 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro7-2956), but the dose-response curve to glucagon was unaltered. Thus, 10(-6) M norepinephrine produced an increase of 40 +/- 5 beats/min in hearts pretreated with Ro7-2956, as compared to an increase of 22 +/- 3 in control hearts (P less than .01). In contrast, 10(-6) M glucagon produced a rate increase of 25 +/- 4 beats/min in treated hearts vs. 26 +/- 4 beats/min in controls. These data are compatible with the hypothesis that adenylate cyclase and cyclic AMP are involved in the chronotropic response of the fetal mouse heart to norepinephrine but not to glucagon.     

Low Serum Dopamine β-Hydroxylase Activity: A MARKER OF CONGESTIVE HEART FAILURE

To gain information about the nature of disturbances in sympathetic nervous system control in congestive heart failure, serum dopamine β-hydroxylase (DBH) activity was measured in 30 patients with heart failure of diverse etiologies and 29 healthy normotensive controls. The heart failure patients had been symptomatic for at least 6 wk and had elevated filling pressures, low cardiac indices, low ejection fractions, and wide arteriovenous oxygen differences. DBH activity was 47.1±4.7 (mean±SE) for the controls and 14.4±2.7 IU for the heart failure patients (P < 0.001). Sera from some patients with heart failure had potent inhibitory effects on DBH activity of normal sera. The inhibitor was heat stable and dialyzable and could be demonstrated despite presence of N-ethylmaleimide or Cu⁺⁺ in the reaction mixture. However, some inhibitory activity was also present in sera of normal patients; this inhibitory property was not demonstrable in unheated normal serum, but was unmasked when DBH was heat inactivated. It is proposed that although the inhibitor may have been a factor in low serum DBH activity in some patients with heart failure, the major cause of the low activity in the heart failure group was a reduced rate of synthesis or release of the enzyme by sympathetic nerves. This may reflect a dissociation between rates of neural release of norepinephrine and release of DBH in chronic, severe heart failure. The observation of low serum DBH levels in patients with heart failure suggests that measurement of DBH levels may serve as a useful indicator of cardiac dysfunction.     

Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient µ_t using optical coherence tomography. The resulting values of the local total attenuation coefficient µ_t (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm⁻¹) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm⁻¹), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm⁻¹) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm⁻¹) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy.OCIS codes: (110.4500) Optical coherence tomography, (170.6935) Tissue characterization, (100.2960) Image analysis, (290.1350) Backscattering     

Calibration of myocardial T2 and T1 against iron concentration

Background

The assessment of myocardial iron using T2* cardiovascular magnetic resonance (CMR) has been validated and calibrated, and is in clinical use. However, there is very limited data assessing the relaxation parameters T1 and T2 for measurement of human myocardial iron.

Methods

Twelve hearts were examined from transfusion-dependent patients: 11 with end-stage heart failure, either following death (n = 7) or cardiac transplantation (n = 4), and 1 heart from a patient who died from a stroke with no cardiac iron loading. Ex-vivo R1 and R2 measurements (R1 = 1/T1 and R2 = 1/T2) at 1.5 Tesla were compared with myocardial iron concentration measured using inductively coupled plasma atomic emission spectroscopy.

Results

From a single myocardial slice in formalin which was repeatedly examined, a modest decrease in T2 was observed with time, from mean (±SD) 23.7 ± 0.93 ms at baseline (13 days after death and formalin fixation) to 18.5 ± 1.41 ms at day 566 (p < 0.001). Raw T2 values were therefore adjusted to correct for this fall over time. Myocardial R2 was correlated with iron concentration [Fe] (R² 0.566, p < 0.001), but the correlation was stronger between LnR2 and Ln[Fe] (R² 0.790, p < 0.001). The relation was [Fe] = 5081•(T2)^-2.22 between T2 (ms) and myocardial iron (mg/g dry weight). Analysis of T1 proved challenging with a dichotomous distribution of T1, with very short T1 (mean 72.3 ± 25.8 ms) that was independent of iron concentration in all hearts stored in formalin for greater than 12 months. In the remaining hearts stored for <10 weeks prior to scanning, LnR1 and iron concentration were correlated but with marked scatter (R² 0.517, p < 0.001). A linear relationship was present between T1 and T2 in the hearts stored for a short period (R² 0.657, p < 0.001).

Conclusion

Myocardial T2 correlates well with myocardial iron concentration, which raises the possibility that T2 may provide additive information to T2* for patients with myocardial siderosis. However, ex-vivo T1 measurements are less reliable due to the severe chemical effects of formalin on T1 shortening, and therefore T1 calibration may only be practical from in-vivo human studies.     

Renin-Angiotensin System Inhibition in Conscious Dogs during Acute Hypoxemia: EFFECTS ON SYSTEMIC HEMODYNAMICS, REGIONAL BLOOD FLOWS, AND TISSUE METABOLISM

The role of the renin-angiotensin system in mediating the circulatory and metabolic responses to hypoxia was studied in three groups of conscious dogs that were infused continuously with normal saline, teprotide (10 μg/kg per min), and saralasin (1 μg/kg per min), respectively. Hypoxia was produced by switching from breathing room air to 5 or 8% oxygen-nitrogen mixture. Plasma renin activity increased from 2.3±0.4 to 4.9±0.8 ng/ml per h during 8% oxygen breathing, and from 2.8±0.4 to 8.4±1.8 ng/ml per h during 5% oxygen breathing. As expected, cardiac output, heart rate, mean aortic blood pressure, and left ventricular dP/dt and dP/dt/P increased during both 5 and 8% oxygen breathing in the saline-treated dogs; greater increases occurred during the more severe hypoxia. Teprotide and saralasin infusion diminished the hemodynamic responses to 5% oxygen breathing, but did not affect the responses to 8% oxygen breathing significantly. In addition, the increased blood flows to the myocardium, kidneys, adrenals, brain, intercostal muscle, and diaphragm that usually occur during 5% oxygen breathing were reduced by both agents. These agents also reduced the increases in plasma norepinephrine concentration during 5% oxygen breathing, but had no effects on tissue aerobic or anaerobic metabolism.     

Assessment of Liver Fibrosis with Diffusion-Weighted Magnetic Resonance Imaging Using Different b-values in Chronic Viral Hepatitis

Objective

To examine the effectiveness of apparent diffusion coefficient (ADC) values and to compare the reliability of different b-values in detecting and identifying significant liver fibrosis.

Subjects and Methods

There were 44 patients with chronic viral hepatitis (CVH) in the study group and 30 healthy participants in the control group. Diffusion-weighted magnetic resonance imaging (DWI) was performed before the liver biopsy in patients with CVH. The values of ADC were measured with 3 different b-values (100, 600, 1,000 s/mm²). In addition, liver fibrosis was classified using the modified Ishak scoring system. Liver fibrosis stages and ADC values were compared using areas under the receiver-operating characteristic (ROC) curve.

Results

The study group''s mean ADC value was not statistically significantly different from the control group''s mean ADC value at b = 100 s/mm² (3.69 ± 0.5 × 10⁻³ vs. 3.7 ± 0.3 × 10⁻³ mm²/s) and b = 600 s/mm² (2.40 ± 0.3 × 10⁻³ vs. 2.5 ± 0.5 × 10⁻³ mm²/s). However, the study group''s mean ADC value (0.99 ± 0.3 × 10⁻³ mm²/s) was significantly lower than that of the control group (1.2 ± 0.1 × 10⁻³ mm²/s) at b = 1,000 s/mm². With b = 1,000 s/mm² and the cutoff ADC value of 0.0011 mm²/s for the diagnosis of liver fibrosis, the mean area under the ROC curve was 0.702 ± 0.07 (p = 0.0015). For b = 1,000 s/mm² and the cutoff ADC value of 0.0011 mm²/s to diagnose significant liver fibrosis (Ishak score = 3), the mean area under the ROC curve was 0.759 ± 0.07 (p = 0.0001).

Conclusion

Measurement of ADC values by DWI was effective in detecting liver fibrosis and accurately identifying significant liver fibrosis when a b-value of 1,000 s/mm² was used.Key Words: Diffusion-weighted imaging, Hepatitis, Fibrosis, Liver     

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.The ability to reprogram cellular metabolism, a hallmark of cancer first noted long ago (Warburg et al., 1927) and recently reappreciated, is essential for tumor progression (Hanahan and Weinberg, 2011). Although cancer-initiated metabolic reprogramming processes are promising therapeutic targets (Vander Heiden, 2011), the existence of alternative, compensatory biosynthetic pathways presents a significant challenge for developing such therapies. For example, in lipid metabolism, cancer cells scavenge extracellular lipids as an alternative to energy-requiring de novo fatty acid biosynthesis (Kamphorst et al., 2011). In amino acid metabolism, glycine and serine required for tumor growth can be produced de novo and can also be scavenged from the extracellular environment (Jain et al., 2012; Maddocks et al., 2013).Nucleotide metabolism also involves redundant and convergent biosynthetic pathways. Deoxyribonucleotide triphosphate (dNTP) pools required for DNA replication and repair can be produced by the de novo pathway (DNP) or by the nucleoside salvage pathway (NSP; Fig. 1 A; Reichard, 1988). The DNP uses glucose and amino acids to generate ribonucleotide diphosphates (NDPs), which are converted to deoxyribonucleotide diphosphates (dNDPs) by ribonucleotide reductase (RNR). The same dNDPs can also be produced via the NSP (Reichard, 1988), starting with extracellular deoxyribonucleosides (dNs) which are imported in the cell via specialized transporters. The first enzymatic steps in the cytosolic NSP are catalyzed by two kinases: thymidine kinase 1 (TK1) phosphorylates thymidine (dT), while deoxycytidine (dC) kinase (dCK) phosphorylates dC, deoxyadenosine (dA), and deoxyguanosine (dG; Reichard, 1988). The relevance of these two NSP kinases for dNTP production in normal and malignant cells is yet to be defined. Because dN substrates for the NSP kinases are absent from most cell culture media, it has been assumed that the NSP is dispensable for DNA replication (Xu et al., 1995). However, recent in vivo findings have challenged this assumption. For example, we reported impaired hematopoiesis in dCK^−/− mice due to dCTP pool deficiency, resulting in replication stress (RS), S-phase arrest, and DNA damage in hematopoietic progenitors (Toy et al., 2010; Austin et al., 2012). Analyses of dCK/TK1 double-knockout mice showed that NSP-derived dCTP synthesis is required to compensate for the inhibition of de novo dCTP production (Austin et al., 2012; Fig. 1 A). The mechanism of DNP inhibition involves allosteric regulation of RNR-mediated reduction of cytidine diphosphate (CDP) to dC diphosphate (dCDP) by dT triphosphate (dTTP) produced via TK1 from endogenous dT (Austin et al., 2012; Fig. 1 A).Open in a separate windowFigure 1.dC salvage via dCK prevents dT-induced lethal RS in T-ALL cells. (A) Allosteric control of DNP dCTP production by dT via dTTP. (B) Effects of dT treatment (24 h) on dCTP and dTTP pools. Values represent mean ± SEM. (C) CEM cell cycle analysis after treatment with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (D) CEM cell cycle analysis after treatment with 50 µM hydroxyurea, 15 µM 5-fluorouracil, or 1.6 µM cisplatin for 24 h ± 2.5 µM dC. (E and F) Representative immunoblots of dCK and actin expression (E) and dCK kinase assay (F) in CEM dCK^wt (scrambled shRNA) cells and dCK^low (shRNA against dCK) cells. Values are mean ± SEM. ***, P < 0.001. (G) dCTP levels in CEM dCK^wt and dCK^low cells treated for 24 h with vehicle or 50 µM dT ± 2.5 µM dC. Values are mean ± SEM. ***, P < 0.001. (H) Cell cycle analysis of CEM dCK^low cells treated with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (I) Representative immunoblots detecting Chk1, pChk1 (Ser345), Chk2, pChk2 (Thr68), dCK, and actin in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24, 48, and 72 h. (J) pH2A.X (Ser139) and DNA content (DAPI) in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24 h. (K) Representative images and quantification of the COMET assay conducted on CEM dCK^wt and dCK^low cells 48 h after treatment with vehicle or 50 µM dT in the presence of 2.5 µM dC. Values represent the mean Olive Tail Moment ± SEM from 100 cells per image × 4 images/group; n = 2 independent experiments. ***, P < 0.001. Bars, 50 µm. (L) Annexin V staining of CEM dCK^wt and dCK^low cells after treatment with vehicle, 2.5 µM dC, 50 µM dT, or dC + dT for 72 h. All values are mean ± SEM from at least three replicates/data point. ***, P < 0.001. All data are representative of n = 3 independent experiments, unless indicated.Production of dNTPs by the NSP may be therapeutically relevant in cancer. For example, the ability of cancer cells to switch their dCTP synthesis from the DNP to the NSP may explain why dT given as a single dCTP-depleting agent showed limited efficacy in clinical trials (Chiuten et al., 1980; Kufe et al., 1980, 1981). If correct, this hypothesis suggests that a combination of dT (to inhibit DNP-mediated dCTP production) and a dCK inhibitor (to co-target dCTP production by the NSP) would be more efficacious in killing tumor cells than either treatment alone. Here, we investigate this possibility in the context of acute lymphoblastic leukemia (ALL). We demonstrate that co-targeting both de novo and salvage pathways for dCTP biosynthesis is well tolerated in mice and is efficacious in T-ALL and B-ALL models. We also describe a positron emission tomography (PET)–based assay to noninvasively monitor in vivo pharmacological targeting of dCTP biosynthesis in cancer cells.     

Quantification of the Glycemic Response to Microdoses of Subcutaneous Glucagon at Varying Insulin Levels

OBJECTIVE

Glucagon delivery in closed-loop control of type 1 diabetes is effective in minimizing hypoglycemia. However, high insulin concentration lowers the hyperglycemic effect of glucagon, and small doses of glucagon in this setting are ineffective. There are no studies clearly defining the relationship between insulin levels, subcutaneous glucagon, and blood glucose.

RESEARCH DESIGN AND METHODS

Using a euglycemic clamp technique in 11 subjects with type 1 diabetes, we examined endogenous glucose production (EGP) of glucagon (25, 75, 125, and 175 μg) at three insulin infusion rates (0.016, 0.032, and 0.05 units/kg/h) in a randomized, crossover study. Infused 6,6-dideuterated glucose was measured every 10 min, and EGP was determined using a validated glucoregulatory model. Area under the curve (AUC) for glucose production was the primary outcome, estimated over 60 min.

RESULTS

At low insulin levels, EGP rose proportionately with glucagon dose, from 5 ± 68 to 112 ± 152 mg/kg (P = 0.038 linear trend), whereas at high levels, there was no increase in glucose output (19 ± 53 to 26 ± 38 mg/kg, P = NS). Peak glucagon serum levels and AUC correlated well with dose (r² = 0.63, P < 0.001), as did insulin levels with insulin infusion rates (r² = 0.59, P < 0.001).

CONCLUSIONS

EGP increases steeply with glucagon doses between 25 and 175 μg at lower insulin infusion rates. However, high insulin infusion rates prevent these doses of glucagon from significantly increasing glucose output and may reduce glucagon effectiveness in preventing hypoglycemia when used in the artificial pancreas.     

Optical Coherence Tomography of the Labial Salivary Glands Reveals Age‐Related Differences in Women

The labial minor salivary glands (LSGs) play a role in medical research and practice due to their superficial location and involvement in both systemic and localized diseases. Swept‐source optical coherence tomography (OCT) is a noninvasive modality that enables in vivo, micrometer resolution, wide‐field three‐dimensional imaging in seconds. A purpose‐built swept‐source OCT instrument was employed to acquire three‐dimensional datasets covering the area of 2.43 cm² of the mucosa of the lower lip to the depth of 3.4 mm in young (n = 14; mean age ± SD: 27 ± 3 years; body mass index [BMI] 20.4 ± 2.3 kg/m²) and middle‐aged women (n = 11; 54 ± 6 years; 25.5 ± 3.2 kg/m²). Glandular tissue reflectivity mode (range 0–255; 86 ± 17 vs. 68 ± 12, p = 0.005), average single LSG area in tissue sample (5.26 ± 2.62 mm² vs. 2.87 ± 1.26 mm², p = 0.011), and LSG surface filling factor (0.23 ± 0.13 vs. 0.11 ± 0.10, p = 0.027) had higher values in younger than in middle‐aged women. A correlation between BMI and glandular tissue reflectivity mode (Spearman''s ρ = –0.60) was found (p = 0.002). The results highlight the potential value of LSGs’ OCT morphometry in research regarding ageing.     

Hyperglucagonemia Does Not Explain the β-Cell Hyperresponsiveness and Insulin Resistance in Dysglycemic Youth Compared With Adults: Lessons From the RISE Study

OBJECTIVETo determine whether β-cell hyperresponsiveness and insulin resistance in youth versus adults in the Restoring Insulin Secretion (RISE) Study are related to increased glucagon release.RESEARCH DESIGN AND METHODSIn 66 youth and 350 adults with impaired glucose tolerance (IGT) or recently diagnosed type 2 diabetes (drug naive), we performed hyperglycemic clamps and oral glucose tolerance tests (OGTTs). From clamps we quantified insulin sensitivity (M/I), plasma fasting glucagon and C-peptide, steady-state glucagon and C-peptide at glucose of 11.1 mmol/L, and arginine-stimulated glucagon (acute glucagon response [AGR]) and C-peptide (ACPRmax) responses at glucose >25 mmol/L.RESULTSMean ± SD fasting glucagon (7.63 ± 3.47 vs. 8.55 ± 4.47 pmol/L; P = 0.063) and steady-state glucagon (2.24 ± 1.46 vs. 2.49 ± 1.96 pmol/L, P = 0.234) were not different in youth and adults, respectively, while AGR was lower in youth (14.1 ± 5.2 vs. 16.8 ± 8.8 pmol/L, P = 0.001). Significant age-group differences in insulin sensitivity, fasting C-peptide, steady-state C-peptide, and ACPRmax were not related to glucagon. Fasting glucose and glucagon were positively correlated in adults (r = 0.133, P = 0.012) and negatively correlated in youth (r = −0.143, P = 0.251). In both age-groups, higher fasting glucagon was associated with higher fasting C-peptide (youth r = 0.209, P = 0.091; adults r = 0.335, P < 0.001) and lower insulin sensitivity (youth r = −0.228, P = 0.066; adults r = −0.324, P < 0.001). With comparable fasting glucagon, youth had greater C-peptide and lower insulin sensitivity. OGTT suppression of glucagon was greater in youth.CONCLUSIONSYouth with IGT or recently diagnosed type 2 diabetes (drug naive) have hyperresponsive β-cells and lower insulin sensitivity, but their glucagon concentrations are not increased compared with those in adults. Thus, α-cell dysfunction does not appear to explain the difference in β-cell function and insulin sensitivity in youth versus adults.     

A high mean arterial pressure target is associated with improved microcirculation in septic shock patients with previous hypertension: a prospective open label study

IntroductionThe effect of mean arterial pressure titration to a higher level on microcirculation in septic shock patients with previous hypertension remains unknown. Our goal is to assess the effect of mean arterial pressure titration to a higher level on microcirculation in hypertensive septic shock patients.MethodsThis is a single-center, open-label study. Hypertensive patients with septic shock for less than 24 hours after adequate fluid resuscitation and requiring norepinephrine to maintain a mean arterial pressure of 65 mmHg were enrolled. Mean arterial pressure was then titrated by norepinephrine from 65 mmHg to the normal level of the patient. In addition to hemodynamic variables, sublingual microcirculation was evaluated by sidestream dark field imaging.ResultsNineteen patients were enrolled in the study. Increasing mean arterial pressure from 65 mmHg to normal levels was associated with increased central venous pressure (from 11 ± 4 to 13 ± 4 mmHg, P = 0.002), cardiac output (from 5.4 ± 1.4 to 6.4 ± 2.1 l/minute, P = 0.001), and central venous oxygen saturation (from 81 ± 7 to 83 ± 7%, P = 0.001). There were significant increases in small perfused vessel density (from 10.96 ± 2.98 to 11.99 ± 2.55 vessels/mm², P = 0.009), proportion of small perfused vessels (from 85 ± 18 to 92 ± 14%, P = 0.002), and small microvascular flow index (from 2.45 ± 0.61 to 2.80 ± 0.68, P = 0.009) when compared with a mean arterial pressure of 65 mmHg.ConclusionsIncreasing mean arterial pressure from 65 mmHg to normal levels is associated with improved microcirculation in hypertensive septic shock patients.

Trial registration

Clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01443494","term_id":"NCT01443494"}}NCT01443494; registered 28 September 2011.

Electronic supplementary material

The online version of this article (doi:10.1186/s13054-015-0866-0) contains supplementary material, which is available to authorized users.     

Epoxy-α-Lapachone Has In Vitro and In Vivo Anti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm²), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm²) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm²). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound''s effects on the parasite.     

Chemopreventive Effect of Ardisia crispa Hexane Fraction on the Peri-Initiation Phase of Mouse Skin Tumorigenesis

Objective

To investigate the chemopreventive effect of the hexane extract of Ardisia crispa during the peri-initiation phase of mouse skin tumorigenesis.

Materials and Methods

This study was conducted for 12 weeks on two-stage 7,12-dimethylbenz(α)-anthracene (DMBA)-induced tumor initiation followed by croton-oil-induced tumor promotion in mice. A. crispa root hexane extract (ACRH) was applied at various doses (30, 100, 300 mg/kg) 7 days prior to and after DMBA treatment. Throughout the study, morphological observations, i.e., tumor incidence, tumor volume and tumor burden were measured for each of the treated groups. At the end of the experiment, the mice were sacrificed and their skin tissues were examined histopathologically.

Results

The highest dose of ACRH (300 mg/kg) significantly delayed tumor formation (week 9, p < 0.05) and exhibited the lowest tumor volume (0.71 ± 0.00 mm³, p < 0.05), tumor burden (2.00 ± 0.00, p < 0.05), and tumor incidence (16.67s%, p < 0.05) compared to other doses of ACRH. A 100-mg/kg dose produced tumor latency at week 7, tumor volume of 2.44 ± 0.88 mm³ (p < 0.05), tumor burden of 1.60 ± 0.60 (p < 0.05), and tumor incidence of 50s%; 30 mg/kg produced tumor latency at week 8, tumor volume of 2.04 ± 0.45 mm³ (p < 0.05), tumor burden of 2.17 ± 0.54, tumor incidence of 60s% and carcinogen control (tumor latency at week 7; tumor volume, 3.56 mm³; tumor incidence of 66.67s%).

Conclusion

The highest dose of A. crispa hexane extract delayed tumor development, thus showing a chemopreventive effect on mouse skin tumorigenesis.Key Words: Ardisia crispa, 7,12-Dimethylbenz(α)-anthracene, Tumor burden, Tumor volume     

Defective cellular immunity in renal failure: depression of reactivity of lymphocytes to phytohemagglutinin by renal failure serum

In defining host resistance factors in uremia, experiments were designed to assess the effect of renal failure serum upon the reactivity of normal human lymphocytes to phytohemagglutinin in vitro. Normal buffy coat cells were resuspended in sera obtained from normal subjects and from 14 patients with renal failure, then stimulated with phytohemagglutinin M and the cellular response measured by the increase in thymidine or uridine uptake. The mean thymidine uptake by stimulated cells in normal sera was 14,389 ±1695 (SEM) cpm per 2 × 10⁶ lymphocytes. Uridine uptake under the same conditions was 12,540 ±1887 cpm. Compared to these are a mean thymidine uptake of 2740 ±457 cpm and uridine uptake of 3928 ±667 cpm in renal failure sera. Both differences are significant at P<0.01 level.