Phosphorylase Enzyme Activity Research Articles

Experiences of living with GSD5 (McArdle) disease: challenges and strategies. A qualitative study in the Netherlands

Purpose Glycogen Storage Disease type 5 (GSD5 or McArdle disease) is caused by deficient glycogen phosphorylase enzyme activity in skeletal muscles. Individuals with GSD5 experience symptoms like muscle pain, fatigue, and tachycardia during exertion. Our study aimed to explore the lived experiences of individuals with GSD5, focusing on their daily challenges, the process of being diagnosed, and management strategies. Methods Participants were invited to share their life experiences through in-depth, semi-structured interviews, and the collected data was analyzed using thematic analysis. Results Using purposeful sampling, 13 individuals with GSD5 were recruited for the study. The analysis identified four key themes: “experiencing incomprehensible difficulties,” “diagnosis as an explanation,” “finding ways to manage daily challenges,” and “listening to your body.” These themes reflect diverse experiences of daily functioning, physical challenges faced, the pivotal role of diagnosis in understanding symptoms, and the adoption of management strategies like using the ‘second wind’ phenomenon. Participants emphasized the importance of support networks and adaptive lifestyle changes in effectively managing their condition. Conclusions Early diagnosis and tailored management strategies are critical for improving outcomes and quality of life in individuals with GSD5. Timely diagnosis and comprehensive multidisciplinary care are essential for effectively managing the complexities of this rare metabolic disorder.

A novel description of the Vastus lateralis morphology of the Temminck's ground pangolin (Manis temminckii).

The pangolin (Manidae family) is the world's most trafficked animal, yet very little is known about its physiology and metabolism primarily due to its inconspicuous and solitary nature. Skeletal muscle samples from the Vastus lateralis were collected postmortem from a single female Temminck's ground pangolin (Manis temminckii). Samples were analyzed for fiber type composition, fiber size and myosin heavy chain isoform content. The oxidative and glycolytic metabolic capacity was determined through citrate synthase, 3-hydroxyacetyl co A dehydrogenase, creatine kinase, lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase enzyme activities. Lastly, antioxidant capacity was determined through superoxide dismutase and catalase enzyme activities, and the total antioxidant capacity. The pangolin metabolic profile was then compared to other endurance and nonendurance mammals, in which data were standardized relative to human endurance athletes in order to provide context. Slow twitch type I fibers, rich in mitochondria were the predominant fiber type within the pangolin indicating a reliance on oxidative derived energy from fats and carbohydrates. This suggests that the pangolin has a high endurance capability when compared to other wild animals and human endurance athletes. This is the first study to investigate the skeletal muscle physiology and metabolism of any pangolin species, in an attempt to further understand this endangered animal and aid with conservation efforts.

The feeding status regulates the action of insulin and 20-hydroxyecdysone on haemolymph trehalose level in Bombyx larvae.

Insulin in mammals is known for its effect on carbohydrate metabolism and maintenance of blood sugar levels. In the present study, we explored the effect of exogenous insulin and 20-hydroxyecdysone (20E) on carbohydrate metabolism in Bombyx mori under the fed and food-deprived conditions. The study showed that insulin and 20E regulate the trehalose (major circulating sugar) level in B. mori, and larval feeding status plays a decisive role in influencing the action of these two hormones. At feeding, both insulin and 20E showed its hypertrehalosemic action but at food deprivation, these hormones acted as hypotrehalosemic factors. Although both insulin and 20E showed the same effect on the haemolymph trehalose level either at feeding or food deprivation, the metabolic regulation was different for these two hormones. Insulin treatment to fed larvae increased the haemolymph trehalose level without altering the effectiveness of trehalose utilization but possibly by inducing the activity of glycogen phosphorylase enzyme and releasing glucose-1-P for the increased synthesis of trehalose. The treatment of 20E to fed larvae also increased the trehalose level, but concurrently it also increased both the enzyme activity of trehalase and glycogen phosphorylase. Insulin treatment to food-deprived larvae decreased the circulating trehalose level by increasing the trehalose breakdown as the mRNA expression level of trehalase-2 and enzyme activity of trehalase increased in these larvae. The treatment of 20E to food-deprived larvae decreased the haemolymph trehalose possibly by decreasing its synthesis, as glycogen phosphorylase enzyme activity decreased in these larvae, thus restricting the availability of glucose-1-P for trehalose synthesis. The study, thus suggests that both insulin and 20E regulate carbohydrate metabolism in B. mori.

Enhanced pollutant removal from rural non-point source wastewater using a two-stage multi-soil-layering system with blended carbon sources: Insights into functional genes, microbial community structure and metabolic function

A two-stage multi-soil-layering system with blended carbon sources (MSL–BCS) was constructed at pilot scale for treatment of rural non-point source wastewater. Results showed the MSL–BCS system had effective removal efficiencies with 64% of TN and 60% of TP, respectively. The addition of BCS could result in higher (1.6–3.1 fold) denitrification gene abundances (nirS and nosZ) for enhancing denitrification. High-throughput sequencing approach revealed that the higher abundance (>50%) of Epsilonbacteraeotra (Genus: Sulfuricurvum, Family: Thiovulaceae, Class: Campylobacteria, Phylum: Epsilonbacteraeota) enriched in the surface of BCS, which suggested that Epsilonbacteraeotra are the keystone species in achieving nitrogen removal through enhancing denitrification at oligotrophic level. KEGG analysis indicated that BCS might release some signaling molecules for enhancing the energy metabolism process, as well as stimulate the enzyme activities of histidine kinase, glycogen phosphorylase and ATPase, and thereby the denitrification processes were strengthened in MSL-BCS system. Consequently, this study could provide some valuable information on the removal performance and mechanism of engineering MSL systems packed with BCS to govern the rural wastewater treatment.

Assessment of pharmacokinetic variations of capecitabine after multiple administration in rats: a physiologically based pharmacokinetic model.

Capecitabine is a prodrug of 5-fluorouracil (5-FU) used for the treatment of colorectal cancer, with a two-week course of administration. However, the variance in plasma concentration and metabolic enzyme activities after multiple administration of capecitabine and its metabolites is unknown. The aim of this study was to identify the variance and predict the plasma concentration profile of capecitabine and its metabolites, using metabolic enzyme activities, to develop a more effective and safer medication. Rats orally received 180mg/kg of capecitabine once a day for two weeks. Blood samples were collected nine times, and plasma concentration was measured on day 1, 7, and 14. The liver and small intestine were removed after blood sampling and were used in vitro to evaluate metabolic enzyme activities of carboxylesterase, cytidine deaminase, and thymidine phosphorylase. A physiologically based pharmacokinetic (PBPK) model was developed using in vitro results. Area under the plasma concentration-time curve from 0h to infinity of 5-FU on day 7 and day 14 was significantly lower than that on day 1. Intrinsic clearance of thymidine phosphorylase in the liver on day 7 and day 14 was 1.4 and 1.3 times lower than that on day 1, respectively. The PBPK model described the observed plasma concentration of capecitabine and its metabolites. The decreased plasma concentration of capecitabine was caused by decreased metabolic enzyme activity. Efficacy can be improved by dose adjustment of capecitabine based on metabolic enzyme activities, using the PBPK model.

Essentiality of the Maltase AmlE in Maltose Utilization and Its Transcriptional Regulation by the Repressor AmlR in the Acarbose-Producing Bacterium Actinoplanes sp. SE50/110.

Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL; Wendler et al., 2015a), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of the CRISPR/Cas9 technology. By growth experiments and functional assays of ΔamlE, we could show that AmlE is essential for the maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ΔamlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ΔamlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.

Defective fasting-induced PKA activation impairs adipose tissue glycogen degradation in obese Zucker rats.

Obesity is associated with development of insulin resistance in adipose tissue (AT). Human obesity has been associated with increased glycogen deposition in adipocytes. Adipocytes synthesise glycogen prior to the formation of lipids. The present study examined adipose glycogen content in obese Zucker rats and the effect of fasting on glycogen-metabolising enzymes. We hypothesised that obesity imposes a blunted response to fasting through impaired activation of glycogen-metabolizing enzymes, which dampens glycogen mobilization in obese Zucker rats. We investigated the effect of 24h fasting on AT glycogen metabolism in12-week old obese Zucker rats. Epididymal fat pads were collected from rats fed ad-libitum and fasted for 24h. Glycogen content, glycogen synthase and phosphorylase enzyme activity, and PKA activity were analysed as well as total and phosphorylated protein content for glycogen-metabolizing enzymes glycogen synthase and phosphorylase, glucose transporter GLUT4, and cAMP-dependent response element binding protein levels. Twelve-week old obese Zucker rats showed increased AT glycogen content (adipose glycogen content [mean ± SD], lean: 3.95 ± 2.78 to 0.75 + 0.69 µg.mg-1; p < 0.005 fed vs fasted, and obese: 5.23 ± 3.38 to 5.019 ± 1.99 µg.mg-1; p = ns fed and fasted and p < 0.005 lean vs obese), and impaired fasting-induced glycogen mobilization following a 24h fast. These defects wereassociated with dysfunctional glycogen-metabolizing enzymes, characterized by: (1) blunted phosphorylation-mediated activation and downregulated protein expression of glycogen phosphorylase, and (2) an impaired phosphorylation-mediated inactivation of glycogen synthase. Furthermore,these defects were related to impaired fasting-induced protein kinase A (PKA) activation. This study provides evidence of a defective glycogen metabolism in the adipose associated with impaired fasting-induced activation of the upstream kinase protein kinase A, which render a converging point to obesity-related primary alterations in carbohydrate and lipid metabolism in the AT.

TRANSIENT T CELL LYMPHOPENIA IN A NEWBORN FROM MATERNAL IMMUNOSUPPRESSIVE MEDICATIONS

Introduction The newborn screen for Severe Combined Immunodeficiency (SCID) utilizes real time quantitative PCR for T cell receptor excision circles (TRECs) and is highly sensitive for detection of SCID, with a negative predictive value of 100%. However, T cell lymphopenia from other primary and secondary causes is also detected, often posing diagnostic and treatment dilemmas. We present the case of a female newborn with transient T cell lymphopenia from maternal medications administered during pregnancy. Case Description The patient was a female child born at 34 weeks via Cesarean section for fetal decelerations. Maternal antenatal steroids were administered and the post-natal course was unremarkable. Two newborn screens resulted with very low TRECs. At 2 weeks of age during immunology clinic visit, the patient's mother was found to have a history of lupus with prior kidney transplant for which she had been on hydroxychloroquine, tacrolimus, azathioprine, and prednisone throughout pregnancy. Family history was negative for immunodeficiency or unexplained deaths. Initial flow cytometry showed severe pan-lymphopenia although normal percentages of naive T cells. Proliferation to mitogens was low/normal. Immunoglobulins A and M were low/undetectable, although IgE was normal. Adenosine deaminase and purine nucleotide phosphorylase enzyme activity were normal. Interestingly, TRECs normalized upon recheck the following week. Flow cytometry completely normalized within 4 months and initial abnormalities were ultimately attributed to maternal immunosuppressive medications, antenatal steroids, prematurity, and peri-natal stress. The patient is currently 4 months old and doing well. Discussion This case demonstrates decreased fetal/neonatal thymic output, thought to be from maternal immunosuppressive medications, which ultimately recovered spontaneously.

Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice.

Chronic ethanol consumption has been shown to significantly decrease hepatic glycogen content; however, the mechanisms responsible for this adverse metabolic effect are unknown. In this study, we examined the impact chronic ethanol consumption has on time-of-day-dependent oscillations (rhythms) in glycogen metabolism processes in the liver. For this, male C57BL/6J mice were fed either a control or ethanol-containing liquid diet for 5 wk, and livers were collected every 4 h for 24 h and analyzed for changes in various genes and proteins involved in hepatic glycogen metabolism. Glycogen displayed a robust diurnal rhythm in the livers of mice fed the control diet, with the peak occurring during the active (dark) period of the day. The diurnal glycogen rhythm was significantly altered in livers of ethanol-fed mice, with the glycogen peak shifted into the inactive (light) period and the overall content of glycogen decreased compared with controls. Chronic ethanol consumption further disrupted diurnal rhythms in gene expression (glycogen synthase 1 and 2, glycogenin, glucokinase, protein targeting to glycogen, and pyruvate kinase), total and phosphorylated glycogen synthase protein, and enzyme activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of glycogen metabolism. In summary, these results show for the first time that chronic ethanol consumption disrupts diurnal rhythms in hepatic glycogen metabolism at the gene and protein level. Chronic ethanol-induced disruption in these daily rhythms likely contributes to glycogen depletion and disruption of hepatic energy homeostasis, a recognized risk factor in the etiology of alcoholic liver disease.

Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

Attenuation of Armanni–Ebstein lesions in a rat model of diabetes by a new anti-fibrotic, anti-inflammatory agent, FT011

A key morphological feature of diabetic nephropathy is the accumulation and deposition of glycogen in renal tubular cells, known as Armanni-Ebstein lesions. While this observation has been consistently reported for many years, the molecular basis of these lesions remains unclear. Using biochemical and histochemical methods, we measured glycogen concentration, glycogen synthase and glycogen phosphorylase enzyme activities, and mRNA expression and protein levels of glycogenin in kidney lysates from control and transgenic (mRen-2)27 rat models of diabetes that had been treated with and without a new anti-fibrotic agent, FT011. Diabetic nephropathy was associated with increased glycogen content, increased glycogen synthase activity and decreased glycogen phosphorylase activity. Glycogenin, the key protein responsible for initiating the synthesis of each glycogen particle, had very high levels in the diabetic kidney together with increased mRNA expression compared with control kidneys. Treatment with FT011 did not change glycogen synthase or glycogen phosphorylase enzyme activities but prevented both glycogenin mRNA synthesis and accumulation of Armanni-Ebstein lesions in the diabetic kidney. Armanni-Ebstein lesions found in diabetic nephropathy are due to aberrant glycogenin protein levels and mRNA expression, providing an explanation for the increased glycogen concentration found within the diabetic kidney. FT011 treatment in diabetic rats reduced glycogenin levels and, subsequently, renal glycogen concentration.

P05.1 Efficacy and tolerability of miglustat in patients with Niemann – Pick C disease

and dysarthria. She had left sided dystonia since 6 years of age. Because of her fatigue she was bound toWheel chair for 4 years. The parents were not consangineous. Her examination revealed that she was cachectic, had total ophthalmoplegia, pitosis, left facial weakness. Tonus was increased on the left extremities, and had rigidity on the left upper extremity, deep tendon reflexes were hypoactive. Muscle strength was 3/5 on lower extremities. Bilateral babinsky was positive. Previously made tests were as follows; brain MRI; showed hyperintensity on basal ganglions especially on putamen and globus pallidus. MRI spectroscopy showed constant lactate peak on thalamus. MELAS; MERRF; NARP mutations were normal. Muscle biopsy was normal, EEG; normal. With the neurological findings she was thought to have MNGIE: BAER revealed bilateral moderate degree sensory-neural deafness, electromyography revealed demyelinating neuropathy on lower extremities, serum lactate level was normal. Thymidine phosphorylase enzyme activity was normal. Since Thymidine phosphorylase was normal she was reassesed and for Coeliac disease anti-tissue transglutaminase IgA (t-TGIgA) and antigliadin IgA and IgG (AGA IgA and IgG) titres were studied and found to be high. Small intestine biopsy revealed gluten-sensitive enteropathy stage III and she was diagnosed as Coeliac disease. After gluten-free diet her neurological findings improved. We presented this case to show the broad spectrum of neurologic findings in Coeliac Disease.

Organ-specific ATPase and phosphorylase enzyme activities in a food fish exposed to a carbamate insecticide and recovery response

The acute effect of carbofuran, a carbamate insecticide, was studied on adenosine triphosphatase enzymes in gill, kidney, liver and muscle tissues of a food fish, Clarias batrachus. Glycogen and glycogen phosphorylase were investigated in gill and kidney only. Thirty-six fish were exposed to sublethal concentration (7.6mg/L) for 6days. After 6days, 18 fish were released into freshwater in order to study the recovery response. Eighteen fish were kept in clean water as control. Tissues were isolated from control, exposed and recovery fish at the end of 1, 3 and 6days and used for the assay of enzymes. Total ATPase was inhibited in kidney and muscle tissues throughout the exposure period, whereas branchial and hepatic tissues showed initial induction followed by inhibition. Na(+)-K(+) ATPase activity was induced in gill till day 3, whereas in other tissues inhibition was throughout the exposure period. Mg(+2) ATPase activity was inhibited in all tissues except liver. When the fish were released into freshwater, liver recovered almost to control values and other tissues showed organ-specific response. Glycogen content of gill increased initially followed by decrease, and in kidney initial decrease was noted. The recovery response was more in kidney than in gill. Induction in the activity of glycogen phosphorylases was observed in kidney, whereas gill tissue showed mixed response. Recovery was not observed in phosphorylases. Thus, the results of the present study demonstrated the acute effect of carbofuran on a food fish and organ-specific recovery response to insecticidal treatment.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.

Purine Nucleoside Phosphorylase Deficiency in a Patient With Spastic Paraplegia and Recurrent Infections

Purine nucleoside phosphorylase deficiency is a rare autosomal recessive immunodeficiency disease. The characteristic features of the disease include severe T cell immune defects with recurrent infections, a failure to thrive, and progressive neurological findings. To date, 35 cases of purine nucleosidase phosphorylase deficiency have been reported worldwide. A 2-year-old female patient was hospitalized due to recurrent infections starting from 6 months and a fever that had continued for a month. The parents were first cousins. Physical examination showed a failure to thrive, herpetic lesions around the lips, painful lesions on the tongue and the buccal mucosa, lung infection, and spastic paraparesis in the lower extremities. She had motor and mental retardation. Laboratory tests revealed lymphopenia; low CD3, CD4, and CD8 counts; normal immunoglobulin levels; low uric acid; and very low purine nucleoside phosphorylase enzyme activity (1.4 nmol/h/mg; normal range, 490-1530). DNA sequencing of the purine nucleosidase phosphorylase gene revealed a missense homozygous mutation, a G to A transition at exon 4 position 64 (349G>A transition), which led to a substitution of alanine by threonine at codon 117 (Ala117Thr). Both parents were heterozygous for the mutation. This is the second purine nucleosidase phosphorylase deficient case to have been presented and carrying this mutation worldwide. Various antibiotics, antifungal drugs, and intravenous immunoglobulin were used to treat the infections during her 3 months. This form of treatment proved to be unresponsive, resulting in her subsequent death at 26 months of age.

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

Defects in purine nucleoside phosphorylase (PNP) enzyme activity result in abnormal nucleoside homeostasis, severe T cell immunodeficiency, neurological dysfunction, and early death. Protein transduction domain (PTD) can transfer molecules into cells and may help restore PNP activity in cases of PNP deficiency. However, long-term use of PTD to replace enzymes in animal models or patients has not previously been described. We fused human PNP to the HIV-TAT PTD and found that the fusion with TAT changed the retention and distribution of PNP in PNP-deficient mice. TAT induced rapid intracellular delivery of PNP into tissues, including the brain, prevented urinary excretion of PNP, and protected PNP from neutralizing antibodies, resulting in significant extension of the enzyme's biological activity in vivo. Frequent TAT-PNP injections in PNP-deficient mice corrected the metabolic disorder and immune defects with no apparent toxicity. TAT-PNP remained effective over 24 weeks of treatment, resulting in continued improvement in immune function and extended survival. Our data demonstrate that TAT changes the properties of PNP in vivo and that long-term intracellular delivery of PNP by TAT corrects PNP deficiency in mice. We provide evidence to promote further use of PTD to treat diseases that require repeated intracellular enzyme or protein delivery.

Comparisons of physiological and biochemical responses between milkfish ( Chanos chanos) and grass carp ( Ctenopharyngodon idella) to cold shock

Temporal changes in physiological parameters and enzyme activities related to carbohydrate metabolism in the milkfish ( Chanos chanos) and the grass carp ( Ctenopharyngodon idella) under cold shock at 15 °C were examined in order to better understand thermal compensation mechanisms of teleosts with different cold tolerances. The plasma glucose, lactate, and lipid contents as well as enzyme activities of citrate synthase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase, phosphorylase a, and lactate dehydrogenase at varying time intervals were monitored. The results show that changes in plasma glucose and lipid contents of the milkfish were more notable than those of the grass carp, but the elevation in plasma lactate contents in the grass carp occurred earlier and was much greater than that in the milkfish. Furthermore, changes in plasma glucose contents in the milkfish were correlated with the enzyme activities of phosphorylase a and fructose-1,6-bisphosphatase, but in the grass carp they were correlated with glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities instead. In addition, the change in lactate dehydrogenase activity in the grass carp was more notable than those of other enzymes, but lactate dehydrogenase activities in the milkfish steadily decreased during cold compensation. The results demonstrate that the two species (milkfish and grass carp) respond quite differently to cold shock, and this may be related to their differences in cold tolerance.

Metabolic characteristics of muscles in the spiny lobster, Jasus edwardsii, and responses to emersion during simulated live transport

The metabolic characteristics of five muscle groups in the spiny lobster Jasus edwardsii were examined in order to compare their anaerobic and oxidative capacities. Enzyme activities of phosphorylase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase were highest in abdominal muscles supporting anaerobic burst activity. Hexokinase, citrate synthase, and HOAD activities in the leg and antennal muscles indicated higher aerobic potential. Arginine kinase activities were high in all muscle groups indicating that muscle phosphagens are an important energy reserve. Arginine phosphate concentrations in 4th periopod and abdominal flexor muscle from lobsters sampled in the field were higher than any values from captive animals, and approximately five times those for ATP. Muscle lactates were high in captive animals. Responses to emersion during simulated live transport appear to exploit the capacity for functional anaerobiosis and further differentiated the muscle groups. Abdominal muscles were especially sensitive and after 24 h showed significant increases in lactate, glucose, ADP, and AMP. ATP levels appeared to be maintained by muscle phosphagens and raised doubts about the efficacy of the adenylate energy charge in evaluating the emersion response. Haemolymph glucose, lactic acid, and ammonia peaked after 24 h emersion and were largely restored following re-immersion. We propose that arginine phosphate concentrations in the 4th periopod are an appropriate index of metabolic stress, and could lead to improved commercial handling protocols.

Dispersion of cell-to-cell uncoupling precedes low K+-induced ventricular fibrillation

We hypothesize that hypokalemia-related electrolyte imbalance linked with abnormal elevation of intracellular free Ca2+ concentration can cause metabolic disturbances and subcellular alterations resulting in intercellular uncoupling, which favor the occurrence of malignant arrhythmias. Langendorff-perfused guinea pig heart (n = 44) was subjected to a standard Tyrode solution (2.8 mmol/l K+) followed by a K+-deficient solution (1.4 mmol/l K+). Bipolar ECG of the left atria and ventricle was continuously monitored and the incidence of ventricular fibrillation was evaluated. Myocardial tissue sampling was performed during stabilization, hypokalemia and at the onset of fibrillation. Enzyme activities of succinic dehydrogenase, glycogen phosphorylase and 5-nucleotidase were determined using in situ catalytic histochemistry. The main gap junction protein, connexin-43, was labeled using mouse monoclonal antibody and FITC conjugated goat antimouse antibody. Ultrastructure was examined by transmission electron microscopy. The free Ca2+ concentration was measured by the indo-1 method in ventricular cell cultures exposed to a K+-free medium. The results showed that sustained ventricular fibrillation appeared within 15-30 min of low K+ perfusion. This was preceded by ectopic activity, episodes of bigeminy and tachycardia. Hypokalemia induced moderate reversible and sporadically irreversible subcellular alterations of cardiomyocytes and impairment of intercellular junctions, which were heterogeneously distributed throughout myocardium. Patchy areas with decreased enzyme activities and diminished immunoreactivity of connexin-43 were found. Furthermore, lack of external K+ was accompanied by an increase of intracellular Ca2+. The prevention of Ca2+ overload by either 1 mmol/l Ni2+ (Na+/Ca2+ inhibitor), 2.5 mmol/l verapamil, 10 mmol/l d-sotalol or 10 mmol/l tedisamil was associated with the protection against fibrillation. The results indicate that hypokalemia induces Ca2+ overload injury and disturbances in intercellular coupling. Dispersion of these changes throughout the myocardium may serve as the basis for microreentry circuits and thus favor fibrillation occurrence.

Structural and Functional Characterization of theDrosophilaGlycogen Phosphorylase Gene

We identified a P element insertional mutant of theDrosophilaglycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen content is increased in flies homozygous for the mutant allele. TheDGPHgene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of theDrosophilaglycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.