The significance of reduced respiratory chain enzyme activities: clinical, biochemical and radiological associations.

BACKGROUND: Mitochondrial diseases are an important group of neurometabolic disorders in children with varied clinical presentations and diagnosis that can be difficult to confirm. AIM: To report the significance of reduced respiratory chain enzyme (RCE) activity in muscle biopsy samples from children. METHODS: Retrospective odds ratio was used to compare clinical and biochemical features, DNA studies, neuroimaging, and muscle biopsies in 18 children with and 48 without reduced RCE activity. RESULTS: Children with reduced RCE activity were significantly more likely to have consanguineous parents, to present with acute encephalopathy and lactic acidaemia and/or within the first year of life; to have an axonal neuropathy, CSF lactate >4 mmol/l; and/or to have signal change in the basal ganglia. There were positive associations with a maternal family history of possible mitochondrial cytopathy; a presentation with failure to thrive and lactic acidaemia, ragged red fibres, reduced fibroblast fatty acid oxidation and with an abnormal allopurinol loading test. There was no association with ophthalmic abnormalities, deafness, epilepsy or myopathy. CONCLUSION: The association of these clinical, biochemical and radiological features with reduced RCE activity suggests a possible causative link.

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

Intrinsic diurnal variations in cardiac metabolism and contractile function.

Diurnal variation of cardiac function in vivo has been attributed primarily to changes in factors such as sympathetic activity. No study has investigated previously the intrinsic properties of the heart throughout the day. We therefore investigated diurnal variations in metabolic flux and contractile function of the isolated working rat heart and how this related to circadian expression of metabolic genes. Contractile performance, carbohydrate oxidation, and oxygen consumption were greatest in the middle of the night, with little variation in fatty acid oxidation. The expression of all metabolic genes investigated (including regulators of carbohydrate utilization, fatty acid oxidation, and mitochondrial function) showed diurnal variation, with a general peak in the night. In contrast, pressure overload-induced cardiac hypertrophy completely abolished this diurnal variation of metabolic gene expression. Thus, over the course of the day, the normal heart anticipates, responds, and adapts to physiological alterations within its environment, a trait that is lost by the hypertrophied heart. We speculate that loss of plasticity of the hypertrophied heart may play a role in the subsequent development of contractile dysfunction.

Calcitonin gene-related peptide is not essential for the development of pressure overload-induced hypertrophy in vivo.

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCbeta and skeletal alpha-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1-2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP.

Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids.

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, an important modulator of fatty acid oxidation. We hypothesized that increased fatty acid availability would increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all significantly increased the plasma concentration of nonesterified fatty acids, with a concomitant increase in both rat heart and skeletal muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression returned to basal levels. Fatty acids are known to activate peroxisome proliferator-activated receptor-alpha (PPARalpha). Specific PPARalpha stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPARgamma agonist, decreased MCD expression. The sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPARalpha expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce the expression of MCD in rat heart and skeletal muscle. Additional posttranscriptional mechanisms regulating MCD activity appear to exist.

Green fluorescent protein as a quantitative tool.

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

Heparin-binding EGF-like growth factor is up-regulated in the obstructed kidney in a cell- and region-specific manner and acts to inhibit apoptosis.

The expression of certain growth factors in the epidermal growth factor (EGF) family is altered in response to renal injury. Recent studies have demonstrated that heparin binding EGF-like growth factor (HB-EGF) expression may be cytoprotective in response to apoptotic signals. The purpose of this study was to investigate the potential role of HB-EGF in the upper urinary tract following unilateral ureteral obstruction. We present evidence that: i) ureteral obstruction induced cell-specific but transient activation of HB-EGF gene expression; ii) HB-EGF expression in renal epithelial cells increased under conditions where mechanical deformation, such as that caused by hydronephrotic distension, induces apoptosis, but HB-EGF expression did not increase in renal pelvis smooth muscle cells under identical conditions; and iii) enforced expression of HB-EGF served to protect renal epithelial cells from stretch-induced apoptosis. These results suggest a potential mechanism by which the kidney protects itself from apoptosis triggered by urinary tract obstruction.

Stimulus history alters behavioral responses of neuronal growth cones.

Generally, it is assumed that growth cones respond to a specific guidance cue with a single, specific, and stereotyped behavior. However, there is evidence to suggest that previous exposure to a given cue might alter subsequent responses to that cue (Snow and Letourneau, 1992; Shirasaki et al., 1998). We therefore tested the hypothesis that growth cone responses to stimuli are dependent on the history of previous stimulation. Growth cones of chick dorsal root ganglion neurons were exposed to well characterized stimuli: (1) contact with a laminin-coated bead, which causes growth cone turning, or (2) electrical stimulation, which causes growth cone collapse. Although the expected behavioral responses were observed after the initial stimulation, strikingly different responses to a subsequent stimulation were observed. Growth cones that had recovered from electrical stimulation-induced collapse rapidly developed insensitivity to a second identical electrical stimulation. Growth cones that previously turned in response to contact with a laminin-coated bead responded to a second bead with a "stall" or cessation in outgrowth. This stimulus history dependence of growth cone behavior could be generalized across dissimilar stimuli: after contact with a laminin-coated bead, growth cones failed to collapse in response to electrical stimulation. The calcium/calmodulin-dependent protein kinase II (CaMKII) was implicated in this history dependence by pharmacological experiments. Together, these results demonstrate that growth cones can alter their behavioral response rapidly to a given stimulus in a manner dependent on previous history and that knowledge of past events in growth cone navigation may be required to predict future growth cone behavior.

Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling.

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.

AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells.

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5' deletion mutants of a promoter-reporter construct containing 1. 7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions -1301 to -881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides -993 to -973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.

Direct in vivo gene transfer to urological organs.

PURPOSE: Patients with urological disorders may benefit from gene based therapy. We investigated the feasibility of delivering exogenous genes into urological tissues in vivo using direct in vivo electrotransfection. MATERIALS AND METHODS: Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection of pGL3/luciferase and beta-galactosidase reporter gene constructs, followed by an electrical pulse ranging from 55 to 115 msec at 100 V. Direct injection of deoxyribonucleic acid without an electrical pulse served as the control. The transfected and nontransfected organs were retrieved and analyzed by luciferase activity assay, histochemical and immunocytochemical staining for beta-galactosidase, and reverse transcription polymerase chain reaction with primers specific for beta-galactosidase messenger ribonucleic acid. RESULTS: There was significant luciferase activity 1, 3 and 5 days after direct in vivo electrotransfection in kidneys and testes, and after 3, 5, 7 and 10 days in bladders. Positive beta-galactosidase enzyme activity and beta-galactosidase immunoreactivity were observed in the transfected renal tubular cells, testicular interstitial and germ cells, and uroepithelial bladder layer. Reverse transcription-polymerase chain reaction products of the transfected organs were noted, indicating the successful transcription of messenger ribonucleic acid. CONCLUSIONS: This study demonstrates that direct in vivo electrotransfection is a feasible method of transient gene delivery into intact urological organs. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically.

The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease.

Thirty-three children ranging from 2 weeks to 12 years of age were selected for allopurinol loading, 16 on the basis of an increased urinary ourotate excretion detected by routine organic acid analysis (group A), and 17 for clinical reasons suggesting a urea cycle defect (group B). The allopurinol load test proved positive in 13 of 16 patients from group A, mean peak orotate 64.0 mumol/mmol creatinine (upper limit of reference range, 13.2) and 11 of 17 patients from group B, mean peak orotate 41.0 mumol/mmol creatinine (upper limit of reference range, 13.2). Thorough investigation of these patients including urinary and plasma amino acid analysis and, in 17 cases, liver biopsy for histology and measurement of ornithine carbamyltransferase (OCT) and carbamyl-phosphate synthetase (CPS) activity failed to identify any evidence of a urea cycle disorder. However, muscle biopsies performed in 11 patients showed some evidence of mitochondrial disease in four cases, two defined on the basis of reduced respiratory chain enzyme activity and two on the basis of mtDNA abnormalities. These findings indicate that an increased excretion of orotate in sick children may not be uncommon and that a positive allopurinol load test result may not indicate a specific inherited urea cycle defect. In addition, these results raise the interesting possibility that defective ureagenesis may be a feature of mitochondrial disease in some individuals.

ATP released from astrocytes mediates glial calcium waves.

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

Insulin does not change the intracellular distribution of hexokinase in rat heart.

Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.

Ischemic preconditioning in rat heart: no correlation between glycogen content and return of function.

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function. Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 x 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points. Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 x 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 x 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power. In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.

An overview of aesthetic surgery of the face.

This article explores the interface between the aesthetic aspects of dentistry and complementary cosmetic surgery of the face. Dentists do not often think of facial cosmetic surgery and plastic surgeons do not often think of aesthetic dentistry as being complementary to their endeavours. This paper is designed to help breach this gap. An outline is given of the various cosmetic surgery procedures used on the face with emphasis on those in the perioral region. Procedures discussed include meloplasty, blepharoplasty, laser resurfacing, liposculpture, lip augmentation, rhinoplasty, chin augmentation, correction of prominent ears, and skin care.

Effects of insulin on glucose uptake by rat hearts during and after coronary flow reduction.

We tested the hypothesis that low-flow ischemia increases glucose uptake and reduces insulin responsiveness. Working hearts from fasted rats were perfused with buffer containing glucose alone or glucose plus a second substrate (lactate, octanoate, or beta-hydroxybutyrate). Rates of glucose uptake were measured by 3H2O production from [2-3H]glucose. After 15 min of perfusion at a physiological workload, hearts were subjected to low-flow ischemia for 45 min, after which they were returned to control conditions for another 30 min. Insulin (1 mU/ml) was added before, during, or after the ischemic period. Cardiac power decreased by 70% with ischemia and returned to preischemic values on reperfusion in all groups. Low-flow ischemia increased lactate production, but the rate of glucose uptake during ischemia increased only when a second substrate was present. Hearts remained insulin responsive under all conditions. Insulin doubled glucose uptake when added under control conditions, during low-flow ischemia, and at the onset of the postischemic period. Insulin also increased net glycogen synthesis in postischemic hearts perfused with glucose and a second substrate. Thus insulin stimulates glucose uptake in normal and ischemic hearts of fasted rats, whereas ischemia stimulates glucose uptake only in the presence of a cosubstrate. The results are consistent with two separate intracellular signaling pathways for hexose transport, one that is sensitive to the metabolic requirements of the heart and another that is sensitive to insulin.

Regulation of exogenous and endogenous glucose metabolism by insulin and acetoacetate in the isolated working rat heart. A three tracer study of glycolysis, glycogen metabolism, and glucose oxidation.

Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.