Chronic allopurinol and adenine therapy in Duchenne muscular dystrophy: effects on muscle function, nucleotide degradation, and muscle ATP and ADP content

Prompted by the controversy on the efficacy of allopurinol in Duchenne muscular dystrophy and by our observations of an abnormal adenine nucleotide turnover in this disease, we conducted an 18 month, double-blind clinical trial with allopurinol and adenine in 14 Duchenne boys paired according to age and functional activity. Detailed clinical evaluation was performed trimonthly. Muscle ATP and ADP content was measured before and after 1 year of treatment. The effect of therapy on adenine nucleotide turnover was determined. No significant difference was observed between the treated and placebo groups, but both showed a significant deterioration (p less than 0.05) in most clinical parameters. Muscle ATP was reduced in Duchenne dystrophy (p less than 0.02) but did not change with therapy, and no correction of the abnormal adenine nucleotide degradation was observed.     

Neuroprotective effect of adenine on purkinje cell survival in rat cerebellar primary cultures

Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism.     

Extracellular purine catabolites and tissue nucleotides and purine catabolites during progression and recovery of ischemia

During progression and recovery of ischemia, extracellular purine catabolites were measured and compared with purine catabolites and adenine nucleotide levels in the tissue. Ischemia was induced by bilateral occlusion of the common carotid arteries in gerbil. Extracellular purine catabolites were sampled by in vivo brain microdialysis technique, and tissue adenine nucleotides and purine catabolites were extracted from in situ frozen and lypholized brain tissue. These metabolites were measured with simple method of isoclatic condition by HPLC system. Ischemia depleted tissue ATP and ADP, whereas AMP and purine catabolites accumulated strikingly. In parallel, extracellular purine catabolites increased as consecutive degradation on the biochemical metabolic pathway in the same animal. Levels of tissue ATP and adenosine normalized in the recovery phase of ischemia immediately. However, extracellular purine catabolites during the recovery are much higher than those during the progression of ischemia. These data indicated extracellular high levels of purine catabolites during the ischemic insult, and especially adenosine as neuromodulator which may turn off spontaneous neuronal activity and exert antiepileptic effects during the recovery rather than the progression of ischemia.     

Quantitative muscle ultrasound is a promising longitudinal follow-up tool in Duchenne muscular dystrophy

Responsive outcome measures are needed to follow the disease status of Duchenne muscular dystrophy (DMD) patients, as new therapeutic approaches become available for affected boys. Quantitative muscle ultrasound (QMUS) is potentially an attractive follow up tool for DMD because it reflects the severity of the dystrophic process without the need for invasive procedures, by quantifying echo intensity (i.e., mean grey level of muscle images) and muscle thickness. We performed a longitudinal follow-up of lower and upper extremity QMUS in 18 DMD patients and compared this with physical functioning in 11 of these patients. QMUS could be performed in every patient, and no patient was subjected to more than a total of 20min of ultrasound scanning time for this study. As expected we found a significant increase of echo intensity with age, reflecting increasing dystrophic muscle changes. This increase was related to ambulatory status, functional grading, muscle strength and motor ability. Our study establishes QMUS as a practical and child-friendly tool for the longitudinal follow up of DMD patients.     

Purine metabolism of erythrocytes in myotonic dystrophy

Summary The uptake and subsequent metabolism of adenine and adenosine was studied with the intact erythrocytes of patients with myotonic dystrophy (MD). Washed erythrocytes of both controls and patients were incubated with ¹⁴C-labeled adenine or adenosine at 20°C for 5 to 120s to characterize the uptake process. No differences in the uptake process of adenine or adenosine were observed between normal and MD erythrocytes. Formation of adenine nucleotides was determined at 37° C after incubation for 30 min. Compared to controls, the incorporation of adenine into adenine nucleotides was 2.6 times higher in MD erythrocytes. This depends mainly on an increase of ATP formation. The mean ratios of ATP: ADP+AMP for normal red cells and MD erythrocytes were 0.92 and 1.39 respectively. No difference was found in the conversion of adenosine to adenine nucleotides. In spite of a normal amount of intracellular ATP a greater demand for ATP exists. This might be due to leakage of adenine nucleotides out of MD erythrocytes.

---

Zusammenfassung Die Aufnahme und der Stoffwechsel von Adenin und Adenosin in Erythrocyten von Patienten mit myotoner Dystrophie (MD) wurde untersucht. Zur Charakterisierung des Aufnahmevorganges wurden gewaschene Erythrocyten von Kontrollpersonen bzw. Patienten mit ¹⁴C-markiertem Adenin oder Adenosin bei 20° C für 5 bis 120s inkubiert. Zwischen den Kontrollen und den Patienten waren keine Unterschiede nachweisbar. Nach 30 min Inkubation bei 37° C wurden die gebildeten Adeninnukleotide bestimmt. Im Vergleich zu den Kontroll-Erythrocyten wurde in den Erythrocyten der MD-Patienten Adenin um den Faktor 2.6 vermehrt in Adeninnukleotide eingebaut, wobei es zu einem Anstieg des Quotienten aus ATP: ADP+AMP von durchschnittlich 0,92 auf 1,39 kam. Adenosin wurde hingegen in den Erythrocyten beider Kollektive in gleicher Weise metabolisiert. Die Ergebnisse weisen auf keine Störung der Purintransportsysteme an Erythrocyten von MD-Patienten, lassen aber trotz normaler intraerythrocytärer ATP-Konzentration auf einen größeren Bedarf an ATP schließen, der auf einen vermehrten Austritt der Adeninnukeotide aus dem MD-Erythrocyten zurückzuführen sein könnte.

     

Abnormalities of platelet adenine nucleotides in patients with myeloproliferative disorders.

Platelet aggregation and adenine nucleotides in platelets have been studied in thirteen patients with myeloproliferative disorders. ADP induced aggregation was abnormal in two patients, but collagen induced aggregation was impaired in 11 patients. The concentrations of ATP and ADP in resting platelets in the patients with abnormal aggregation were significantly less than those in normal subjects. Marked reduction of the amounts of both nucleotides released into plasma was also observed after stimulation of collagen in these patients. Platelets in the patients with normal functions contained almost normal amounts of adenine nucleotides. We discussed the relationship between platelet dysfunction and adenine nucleotides in platelets of myeloproliferative disorders and concluded that platelet dysfunction was mainly attributable to reduction of releasable ADP.     

Impaired cortical energy metabolism but not major antioxidant defenses in experimental bacterial meningitis

The loss of soluble brain antioxidants and protective effects of radical scavengers implicate reactive oxygen species in cortical neuronal injury caused by bacterial meningitis. However, the lack of significant oxidative damage in cortex [J. Neuropathol. Exp. Neurol. 61 (2002) 605-613] suggests that cortical neuronal injury may not be due to excessive parenchymal oxidant production. To see whether this tissue region exhibits a prooxidant state in bacterial meningitis, we examined the state of the major cortical antioxidant defenses in infant rats infected with Streptococcus pneumoniae. Adenine nucleotides were co-determined to assess possible changes in energy metabolism. Arguing against heightened parenchymal oxidant production, the high NADPH/NADP(+) ratio ( approximately 3:1) and activities of the major antioxidant defense and pentose phosphate pathway enzymes remained unchanged at the time of fulminant meningitis. In contrast, cortical ATP, ADP and total adenine nucleotides were on average decreased by approximately 25%. However, energy depletion did not lead to a significant decrease in adenylate energy charge (AEC). ATP depletion was likely a consequence of metabolic degradation, since it correlated with both the loss of total adenine nucleotides and accumulation of purine degradation products. Furthermore, the loss of ATP and decrease in AEC correlated significantly with the extent of neuronal injury. These results strongly suggest that energy depletion rather than parenchymal oxidative damage is involved in the observed cortical neuronal injury.     

Release of endogenous and radioactive purines from the rabbit retina

The adenine nucleotide pool of rabbit retina was labeled by an intravitreal injection in vivo of [3H]adenosine. Practically all the radioactivity was retained in the form of adenine nucleotides. The relative proportion of [3H]adenine nucleotides was the same as that of endogenous nucleotides. Potassium depolarization (43.6 mM) in vitro caused a rapid increase in the rate of release of radioactive purines. The radioactive material was composed of hypoxanthine, xanthine, inosine and trace amounts of adenine, adenosine and adenine nucleotides. The release of radioactive purines was delayed and reduced by the addition of the nucleoside inhibitor dipyridamole suggesting that the purines may be released in the form of nucleosides. Similarly, the addition of the ecto 5'-nucleotidase inhibitor alpha, beta-methylene ADP (AOPCP) did not alter the release of radioactivity or the composition of the released purines. Endogenous hypoxanthine, xanthine and inosine could be detected in the effluents, but there was only a very modest increase following potassium depolarization. There was a slight, but significant, decrease in the release of endogenous adenosine and increase in AMP after AOPCP. It is concluded that there is an intensive uptake and phosphorylation of adenosine in the rabbit retina. Depolarization induces release of radioactive purine nucleosides and bases. Most of these compounds appear to be released as such, but in addition there may be a small (maximally a few per cent of the total) fraction of the purines that are released as nucleotides.     

Nitric oxide interacts with oxygen free radicals to evoke the release of adenosine and adenine nucleotides from rat hippocampal slices

The present study examined some possible mechanisms underlying the previously demonstrated release of adenosine by nitric oxide (NO) donors. Perfusion with the NO-donor S-nitroso-N-acetyl penicillamine (SNAP; 300 microM) led to a significant increase in the release of [3H]purines from both unstimulated and electrically stimulated hippocampal slices prelabeled with [3H]adenine. The NO-donor also evoked the release of endogenous ATP and ADP from unstimulated slices and, when combined with electrical stimulation, the release of ATP, AMP and adenosine. The SNAP-induced [3H]purine release was calcium-dependent, but not affected by the glutamate receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclohepten-5,10-imine;100 nM) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 10 microM). Zaprinast (5 microM), an inhibitor of the cyclic GMP-dependent phosphodiesterase and 8-Br-cyclic GMP (0.01-1 mM) failed to evoke the release of purines, whereas generation of oxygen free radicals by xanthine plus xanthine oxidase did evoke purine release. Coperfusion of SNAP with the free radical scavengers superoxide dismutase (SOD; 60 microg/ml) and catalase (50 microg/ml) reduced or eliminated the ability of the NO-donor to enhance [3H]purine release, but the poly (ADP-ribosyl) synthetase (PARS) inhibitor benzamide (500 microM) did not affect it. These data indicate that NO interacts with superoxide, likely forming peroxynitrite, which subsequently acts to release adenosine and adenine nucleotides from hippocampal tissue.     

Potassium-stimulated purine release by cultured sympathetic neurons

Environmental factors can influence cultured sympathetic neurons to acquire several different neurotransmitter phenotypes. Cholinergic and noradrenergic transmitter status can be influenced by heart cell conditioned medium, chronic depolarization (Patterson, P. H. (1978) Annu. Rev. Neurosci. 1:1-17), and rat serum (Wolinsky, E. J., and P. H. Patterson, (1985) J. Neurosci. 5:1509-1512); formation of electrical synapses can be induced by insulin (Wolinsky, E. J., H. Patterson, and A. L. Willard (1985) J. Neurosci., 5:1675-1679). Purine release has also been proposed as a possible transmission mode for sympathetic neurons (Potter, D. D., E. J. Furshpan, and S. C. Landis (1983) Fred. Proc. 42:1626-1632), and as such, it is another candidate for environmental modulation. In this report, we assess the ability of sympathetic neuron cultures grown with and without serum to release metabolically labeled tritriated purine compounds in response to depolarization. Exposure to 54 mM potassium stimulated release of adenosine, inosine, and hypoxanthine from both serum-supplemented and defined-medium cultures. However, depolarization-stimulated release of adenine nucleotides was observed only from serum-supplemented cultures and not from serum-free cultures. The release of adenine nucleotides from serum-containing cultures is affected by divalent cations in the manner expected for a neurosecretory process. The failure of serum-free cultures to release detectable adenine nucleotides raises the possibility that they do not share with, or that they differ from, serum-supplemented cultures in the purinergic aspect of the multiple transmission modes available to sympathetic neurons, and that this difference may be due to effects of the culture medium.     

Ovariectomy and estradiol replacement therapy alters the adenine nucleotide hydrolysis in rat blood serum

The low prevalence of coronary heart disease in premenopausal women and its increase after menopause are well established. Many studies have suggested that steroid hormones can inhibit platelet aggregation, reducing the cardiovascular risk. In addition, a number of studies have shown an effect of estrogen on vascular function. The process of haemostasis and thrombus formation can be also affected by adenine nucleotides and adenosine. Consequently, the regulation of enzymes that hydrolyze these nucleotides in the bloodstream is essential in the modulation of the processes of platelet aggregation, vasodilatation and coronary flow. Thus, in this study, we examined the effect of ovariectomy (OVX), estradiol replacement therapy and the in vitro administration of 17beta-estradiol, dehydroisoandrosterone 3-sulfate (DHEAS) and pregnenolone (PREG) on the activity of the enzymes that degrade adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in the blood serum of female rats. OVX significantly increased the hydrolysis of ATP, ADP and AMP, whilst phosphodiesterase activity was unchanged. Estradiol replacement therapy significantly decreased the hydrolysis of the adenine nucleotides and of the substrate marker of phosphodiesterase. In vitro, the addition of steroid hormones did not have any effect on the nucleotide hydrolysis by rat serum. These results suggest the presence of a strong relation between these enzymes and the hormonal system. In addition, the alterations observed are important, because these enzymes control the nucleotides/nucleosides ratio in the circulation and thus the events related to haemostasis.     

Stimulation of reactive astrogliosis in vivo by extracellular adenosine diphosphate or an adenosine A2 receptor agonist

Adenosine and its nucleotides adenosine triphosphate (ATP) and adenosine diphosphate (ADP) stimulate the proliferation of brain astrocytes in vitro and augment the effects of other growth factors. Following brain injury, hypoxia, or around solid tumors with necrotic centers, such as glioblastoma multiformes, high concentrations of adenine nucleotides and adenosine are released into the extracellular space; extracellular adenosine concentrations can rise 30–100-fold to a concentration in excess of 100 μM. Increased concentrations of extracellular adenosine and adenine nucleotides may contribute to reactive astrocytic proliferation following brain injury. To test this hypothesis, adenosine, an adenosine analog 5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA), or ADP was microinjected into rat cortex. The number of glial fibrillary acidic prtein-immunopositive cells was compared between the treated and contralateral saline-injected hemispheres. Within 48 hr, astrocyte density around the CPCA (100 μM) infusion site was almost double that around the control saline infusion site. In hemispheres into which CPCA was infused, there was an increase in astrocytes in the subpial region along fiber tracts and around blood vessels, characteristic of Scherer's secondary structures found in association with malignant astrocytic brain tumors. The increased astrogliosis elicited by CPCA was abolished by coinfusion of the adenosine A₂ receptor antagonist 1,3-dipropyl-7-methylxanthine (DPMX). While microinjection of adenosine (1 mM) failed to stimulate astrogliosis, microinjection of ADP (500 μM) also resulted in a significant reactive astrogliosis and accumulation of astrocytes similar to Scherer's secondary structures. These data indicate that purine nucleosides and nucleotides may play a role in reactive astrogliosis following injury and may also play a role in stimulating the astrocyte migration responsible for Scherer's secondary structures around astrogliomas. © 1994 Wiley-Liss, Inc.     

Exon‐skipping therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the DMD gene for which no mutation‐targeted therapy has been available thus far. However, exon‐skipping mediated by antisense oligonucleotides (AOs), which are short single‐strand DNAs, has considerable potential for DMD therapy, and clinical trials in DMD patients are currently underway. This exon‐skipping therapy changes an out‐of‐frame mutation into an in‐frame mutation, aiming at conversion of a severe DMD phenotype into a mild phenotype by restoration of truncated dystrophin expression. Recently, stable and less‐toxic AOs have been developed, and their higher efficacy was confirmed in mice and dog models of DMD. In this review, we briefly summarize the genetic basis of DMD and the potential and perspectives of exon skipping as a promising therapy for this disease.     

Developmental changes in purine nucleotide metabolism in cultured rat astroglia

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP-5′-nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter.

The results indicate that purine nucleotide metabolism in the cultured astroglia is changing with aging to allow the cells to maintain their nucleotide pool by reutilization of preformed hypoxanthine, rather than by de-novo production of new purines. Aging is also associated with increased capacity for operation of the adenine nucleotide cycle, contributing to the homeostasis of adenine nucleotides and to the energy charge of the cells. In principle, the age-related alterations in purine metabolism in the astroglia resemble those occurring in the maturating neurons, except for the capacity to produce purines de novo, which exhibited inverse trends in the two tissues. However, in comparison to the neurons, the cultured astroglia possess the capacity for a more intensive metabolism of purine nucleotides.     

Valley sign in Becker muscular dystrophy and outliers of Duchenne and Becker muscular dystrophy

Valley sign has been described in patients with Duchenne muscular dystrophy (DMD). As there are genetic and clinical similarities between DMD and Becker muscular dystrophy (BMD), this clinical sign is evaluated in this study in BMD and DMD/BMD outliers. To evaluate the sign, 28 patients with Becker muscular dystrophy (BMD), 8 DMD/BMD outliers and 44 age-matched male controls with other neuromuscular diseases were studied. The sign was examined after asking patients to abduct their arms to about 90 cent with hands directed upwards; the muscle bulk over the back of the shoulders was observed. The sign was considered positive if the infraspinatus and deltoid muscles were enlarged and between these two muscles, the muscles forming the posterior axillary fold were wasted as if there were a valley between the two mounts. Twenty-five BMD patients and 7 DMD/BMD outliers had positive valley sign. However, it was less remarkable in comparison to DMD. It was absent in all the 44 controls. It was concluded that the presence of valley sign may help in differentiating BMD from other progressive neuromuscular disorders of that age group.     

Purinergic Modulation of the Evoked Release of [3H]Acetylcholine from the Hippocampus and Cerebral Cortex of the Rat: Role of the Ectonucleotidases

Modulation by exogenous and endogenous adenine nucleotides and adenosine of [³H]acetylcholine release evoked by veratridine (10 μM) was compared in synaptosomal fractions from the hippocampus and the cerebral cortex of the rat. In both brain areas, exogenously added ATP or adenosine (10–100 μM) inhibited the evoked tritium release. In the hippocampus, ATPμS, an ATP analogue more resistant to catabolism than ATP, was virtually devoid of effect on tritium release, and the effect of ATP was prevented by the ecto-5′-nucleotidase inhibitor α,β-methylene ADP (100 μM), by adenosine deaminase (2 U/ml) and by the A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 20 nM). In contrast, in the cerebral cortex, the effect of ATP on tritium release was not prevented by either α,β-methylene ADP (100 μM) or adenosine deaminase (2 U/ml), and several ATP analogues (30 μM) inhibited release. The order of intensity of the inhibitory effects of the ATP analogues was: ATPγS > ATP > ñ,γ-imido ATP > β,γ-methylene ATP > 2-methyl-S-ATP, α,β-methylene ATP. The effect of ATPγS in the cerebral cortex was prevented by DPCPX (20 nM) and was not affected by the P₂ purinoceptor antagonist suramin (100 μM). In the hippocampus, α,β-methylene ADP (100 μM) increased the evoked release of tritium, and adenosine deaminase (2 U/ml) produced an even greater increase; when adenosine deaminase was added in the presence of α,β-methylene ADP, adenosine deaminase still increased the evoked release of tritium. In the cerebral cortex, DPCPX (20 nM) and adenosine deaminase (2 U/ml) increased the evoked tritium release by a similar magnitude, but the effect of adenosine deaminase was smaller than in the hippocampus. It is concluded that in the cerebral cortex ATP as such presynaptically inhibits acetylcholine release, whereas in the hippocampus the role of adenine nucleotides is as a source of endogenous extracellular adenosine that tonically inhibits acetylcholine release. The results also show that besides formation of adenosine from adenine nucleotides, released adenosine as such contributes in nearly equal amounts to the pool of endogenous adenosine that presynaptically inhibits acetylcholine release in the hippocampus.     

Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle.

Despite promoter tissue specificity, up-regulation of the brain and Purkinje cell type dystrophin isoforms was described in skeletal muscle of X-linked dilated cardiomyopathy (XLDCM) and BMD affected individuals. An extended population of 11 Duchenne muscular dystrophy (DMD) and 11 Becker muscular dystrophy (BMD) patients was investigated to determine whether ectopic muscle expression of the two full-length non-muscular isoforms is a common event in dystrophinopathies and if it has functional significance. Up-regulation of the two non-muscle-specific isoforms was detected in four DMD patients but in none of the BMD affected individuals or non-dystrophic controls. This is the first report of an expression of these two isoforms in DMD skeletal muscle. Ectopic expression is not confined to regenerating or revertant fibers and does not correlate with age at biopsy, clinical phenotype, cardiac involvement, deletion size or location.We consider that muscle ectopic expression of the brain and Purkinje cell-type isoforms has no favorable prognostic significance in DMD and BMD patients.     

Beneficial antipsychotic effects of allopurinol as add-on therapy for schizophrenia: a double blind, randomized and placebo controlled trial

There is a large amount of data showing that adenosine plays a role opposite to dopamine in the brain. Adenosine agonists and antagonists produce behavioral effects similar to dopamine antagonists and dopamine agonists, respectively. Allopurinol, a well-known hypouricemic drug that inhibits xantine oxidase, has been used as an add-on drug in the treatment of poorly responsive schizophrenic patients. Indeed, the neuropsychiatric effects of allopurinol in schizophrenia have been suggested to be secondary to its inhibitory effect of purine degradation, enhancing adenosinergic activity. The purpose of the present investigation was to assess the efficacy of allopurinol as an adjuvant agent in the treatment of chronic schizophrenia in an 8-week double blind and placebo controlled trial. Eligible participations in the study were 46 patients with schizophrenia. All patients were inpatients and were in the active phase of the illness, and met DSM-IV criteria for chronic schizophrenia. Patients were allocated in a random fashion, 23 to haloperidol 15 mg/day plus allopurinol 300 mg/day and 23 to haloperidol 15 mg/day plus placebo. Although both protocols significantly decreased the score of the positive, negative and general psychopathological symptoms over the trial period, the combination of haloperidol and allopurinol showed a significant superiority over haloperidol alone in the treatment of positive symptoms, general psychopathology symptoms as well as PANSS total scores. The means of Extrapyramidal Symptoms Rating Scale for the placebo group were higher than in the allopurinol group over the trial, and the differences were significant in weeks 6 and 8. A significant difference was observed between the overall mean biperiden dosages in two groups. The results of this study suggest that allopurinol may be an effective adjuvant agent in the management of patients with chronic schizophrenia. Nevertheless, results of larger controlled trials are needed, before recommendations for a broad clinical application can be made.     

Ultrasound imaging of muscles in Duchenne muscular dystrophy

The ultrasound imaging of quadriceps, gastrocnemius and soleus muscles was performed in 30 patients with Duchenne muscular dystrophy (DMD) and 16 control subjects. In controls, the skeletal muscle itself was scarcely echogenic. However, bone surface and fascia were clearly echogenic. The transverse scan of muscle in all DMD patients showed an increased echogenicity, which made the echo from bone or fascia less intense. The abnormal muscle echo was graded according to Heckmatt et al. From the quantitative aspect, there was a significant correlation between disability scale of DMD patients and abnormal echogenicity of the quadriceps muscle. A similar correlation was also observed between results of manual muscle testing the ultrasound imaging. The soleus muscle was usually less abnormal than the gastrocnemius in the ultrasound imaging. Thus, the ultrasound imaging seemed to provide an important information for the clinical assessment of DMD patients.     

Levels of adenosine and adenine nucleotides in slices of rat hippocampus

ATP, ADP, AMP, IMP, adenosine, inosine and cyclic AMP were measured in slices of the rat hippocampus maintained in vitro. Immediately following cutting ATP was low (3.5 +/- 0.6 nmol/mg protein) and AMP high (8.6 +/- 0.9 nmol/mg), giving an energy charge of only 0.34 +/- 0.02. Over the next 90 min the energy charge gradually normalized (to 0.92 +/- 0.01), partly due to conversion of AMP to ATP, but mainly to breakdown to adenosine and other purines which were recovered in the incubation medium. Total purine content decreased from approximately 18 to 10 nmol/mg protein in the first hour following cutting. In slices from old rats the energy charge was lower 60 min following preparation than in younger rats, while cyclic AMP and adenosine levels were higher. The adenosine antagonist 8-phenyl-theophylline tended to enhance the recovery of responsiveness after preparation of the slices. Stimulation of excitatory afferent fibers at a frequency of 10 Hz for 5 min did not significantly alter the purine levels in brain slices, while hypoxia decreased the energy charge significantly and tended to increase adenosine levels. These changes occurred somewhat later than the fall in electrophysiological responsiveness. 8-Phenyl-theophylline was able to delay somewhat the decline in the amplitude of synaptic responses under hypoxic conditions. It is concluded that the viable part of the hippocampal slice, which accounts for about half of the tissue, has levels of adenine nucleotides and adenosine which are similar to those found in the intact rat brain. The return of electrophysiological function following slice preparation is paralleled by a normalization of the energy charge, the adenosine level and the concentration of cyclic AMP. The absence of electrophysiological activity following cutting, and the decreases in such responses following either prolonged afferent stimulation or hypoxia may be related to changes in purine concentration in the slice. Although adenosine accumulating in the slice may contribute to the depression of electrophysiological responses it is probably not the major factor responsible for the reduction in synaptic responsiveness.