Distinctive clinical presentation and pathogenic specificities of anti-AK5 encephalitis.

Limbic encephalitis with antibodies against adenylate kinase 5 (AK5) has been difficult to characterize because of its rarity. In this study, we identified 10 new cases and reviewed 16 previously reported patients, investigating clinical features, IgG subclasses, human leucocyte antigen and CSF proteomic profiles. Patients with anti-AK5 limbic encephalitis were mostly male (20/26, 76.9%) with a median age of 66 years (range 48-94). The predominant symptom was severe episodic amnesia in all patients, and this was frequently associated with depression (17/25, 68.0%). Weight loss, asthenia and anorexia were also highly characteristic, being present in 11/25 (44.0%) patients. Although epilepsy was always lacking at disease onset, seizures developed later in a subset of patients (4/25, 16.0%). All patients presented CSF abnormalities, such as pleocytosis (18/25, 72.0%), oligoclonal bands (18/25, 72.0%) and increased Tau (11/14, 78.6%). Temporal lobe hyperintensities were almost always present at disease onset (23/26, 88.5%), evolving nearly invariably towards severe atrophy in subsequent MRIs (17/19, 89.5%). This finding was in line with a poor response to immunotherapy, with only 5/25 (20.0%) patients responding. IgG1 was the predominant subclass, being the most frequently detected and the one with the highest titres in nine CSF-serum paired samples. A temporal biopsy from one of our new cases showed massive lymphocytic infiltrates dominated by both CD4+ and CT8+ T cells, intense granzyme B expression and abundant macrophages/microglia. Human leucocyte antigen (HLA) analysis in 11 patients showed a striking association with HLA-B*08:01 [7/11, 63.6%; odds ratio (OR) = 13.4, 95% confidence interval (CI): 3.8-47.4], C*07:01 (8/11, 72.7%; OR = 11.0, 95% CI: 2.9-42.5), DRB1*03:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7), DQB1*02:01 (8/11, 72.7%; OR = 13.5, 95% CI: 3.5-52.0) and DQA1*05:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7) alleles, which formed the extended haplotype B8-C7-DR3-DQ2 in 6/11 (54.5%) patients (OR = 16.5, 95% CI: 4.8-57.1). Finally, we compared the CSF proteomic profile of five anti-AK5 patients with that of 40 control subjects and 10 cases with other more common non-paraneoplastic limbic encephalitis (five with antibodies against leucine-rich glioma inactivated 1 and five against contactin-associated protein-like 2), as well as 10 cases with paraneoplastic neurological syndromes (five with antibodies against Yo and five against Ma2). These comparisons revealed 31 and seven significantly upregulated proteins in anti-AK5 limbic encephalitis, respectively mapping to apoptosis pathways and innate/adaptive immune responses. These findings suggest that the clinical manifestations of anti-AK5 limbic encephalitis result from a distinct T cell-mediated pathogenesis, with major cytotoxicity-induced apoptosis leading to a prompt and aggressive neuronal loss, likely explaining the poor prognosis and response to immunotherapy.

Involvement of the phosphoryl transfer network on cardiac energetic metabolism during Staphylococcus aureus infection and its association to disease pathophysiology.

Evidences have suggested that the phosphoryl transfer network by the enzymatic activities of creatine kinase (CK), adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), shows new perspectives to understand some disturbances in the energy metabolism during bacterial infections. Thus, the aim of this study was to evaluate whether Staphylococcus aureus infection in mice could alter serum and cardiac activities of these enzymes and their association to disease pathophysiology. For that, we measured total leukocytes, lymphocytes and neutrophils (just 48 h of infection) that were lower in infected animals after 48 and 72 h in infected mice compared with negative control, while total protein and globulin plasma levels were higher after 72 h of infection. The serum CK activity was higher in infected animals 48 and 72 h post-infection compared to the control group, as well as observed for mitochondrial cardiac CK activity. The serum PK activity was higher in infected animals after 72 h of infection compared to the control group, and lower in the cardiac tissue. The cardiac AK activity was lower in infected animals 48 h and 72 h post-infection compared to the control group, while serum and cardiac LDH activities were higher. Based on these evidences, it is possible to conclude that the stimulation of CK activity exerts a key role as an attempt to maintain the bioenergetic homeostasis by the production of phosphocreatine to avoid a rapid fall on the concentrations of total adenosine triphosphate. In summary, the phosphoryl transfer network can be considered a pathway involved in the improvement on tissue and cellular energy homeostasis of S. aureus-infected mice.

Regulators of blood lipids and lipoproteins? PPARδ and AMPK, induced by exercise, are correlated with lipids and lipoproteins in overweight/obese men and women.

PPARδ is a transcription factor regulating the expression of genes involved in oxidative metabolism, which may regulate blood cholesterols through transcription of oxidative and lipoprotein metabolism genes. To determine the association of skeletal muscle PPARδ content with blood lipids and lipoproteins before and following exercise, overweight and obese men (n = 9) and women (n = 7) were recruited; age, BMI, body fat percentage, and Vo(2max) were (means ± SE) 45 ± 2.5 yr, 31.9 ± 1.4 kg/m(-2), 41.1 ± 1.5%, and 26.0 ± 1.3 mLO(2)·kg(-1)·min(-1), respectively. Subjects performed 12 wk of endurance exercise training (3 sessions/wk, progressing to 500 kcal/session). To assess the acute exercise response, subjects performed a single exercise session on a treadmill (70% Vo(2max), 400 kcal energy expenditure) before and after training. Muscle and blood samples were obtained prior to any exercise and 24 h after each acute exercise session. Muscle was analyzed for protein content of PPARδ, PPARα, PGC-1α, AMPKα, and the oxidative and lipoprotein markers FAT/CD36, CPT I, COX-IV, LPL, F(1) ATPase, ABCAI, and LDL receptor. Blood was assessed for lipids and lipoproteins. Repeated-measures ANOVA revealed no influence of sex on measured outcomes. PPARδ, PGC-1α, FAT/CD36, and LPL content were enhanced following acute exercise, whereas PPARα, AMPKα, CPT I, and COX-IV content were enhanced only after exercise training. PPARδ content negatively correlated with total and LDL cholesterol concentrations primarily in the untrained condition (r ≤ -0.4946, P < 0.05), whereas AMPKα was positively correlated with HDL cholesterol concentrations regardless of exercise (r ≥ 0.5543, P < 0.05). Our findings demonstrate exercise-induced expression of skeletal muscle PPARs and their target proteins, and this expression is associated with improved blood lipids and lipoproteins in obese adults.

Adenylate kinase 7 is a prognostic indicator of overall survival in ovarian cancer.

ABSTRACT: Ovarian cancer (OC), a common malignant heterogeneous gynecological tumor, is the primary cause of cancer-related death in women worldwide. Adenylate kinase (AK) 7 belongs to the adenylate kinase (AK) family and is a cytosolic isoform of AK. Recent studies have demonstrated that AK7 is expressed in several human diseases, including cancer. However, there is a scarcity of reports on the relationship between AK7 and OC. Here, we compared the expression of AK7 in normal and cancerous ovarian tissues from The Cancer Genome Atlas database and used the c2 test to assess the correlation between AK7 levels and the clinical symptoms of OC. Finally, the prognostic significance of AK7 in OC was determined using the Kaplan-Meier analyses and Cox regression and performed gene set enrichment analysis to detect any relevant signaling pathways. We found that AK7 levels were substantially downregulated in OC than that in normal ovarian tissues (P < .001). Low AK7 levels were related to the patients' age (P = .0093) in OC. The median overall survival (OS) of patients with low AK7-expressing OC was shorter than patients with high AK7-expressing OC (P = .019). The Cox regression analysis (multivariate) identified low AK7 levels were independently related to the prognosis of OC (HR 1.34; P = .048). Our study demonstrated that the downregulated levels of AK7 could serve as an independent prognostic indicator for the OS in OC. Additionally, gene set enrichment analysis revealed that EMT, apical junction, TGF-b signaling, UV response, and myogenesis were associated in the low AK7 expression phenotype (NOM P < .05).

Red cell adenylate kinase deficiency in India: identification of two novel missense mutations (c.71A>G and c.413G>A).

Adenylate kinase (AK) deficiency is a rare erythroenzymopathy associated with hereditary nonspherocytic haemolytic anaemia along with mental/psychomotor retardation in few cases. Diagnosis of AK deficiency depends on the decreased level of enzyme activity in red cell and identification of a mutation in the AK1 gene. Until, only eight mutations causing AK deficiency have been reported in the literature. We are reporting two novel missense mutation (c.71A > G and c.413G > A) detected in the AK1 gene by next-generation sequencing (NGS) in a 6-year-old male child from India. Red cell AK enzyme activity was found to be 30% normal. We have screened a total of 32 family members of the patient and showed reduced red cell enzyme activity and confirm mutations by Sanger's sequencing. On the basis of Sanger sequencing, we suggest that the proband has inherited a mutation in AK1 gene exon 4 c.71A > G (p.Gln24Arg) from paternal family and exon 6 c.413G > A (p.Arg138His) from maternal family. Bioinformatics tools, such as SIFT, Polymorphism Phenotyping v.2, Mutation Taster, MutPred, also confirmed the deleterious effect of both the mutations. Molecular modelling suggests that the structural changes induced by p.Gln24Arg and p.Arg138His are pathogenic variants having a direct impact on the structural arrangement of the region close to the active site of the enzyme. In conclusion, NGS will be the best solution for diagnosis of very rare disorders leading to better management of the disease. This is the first report of the red cell AK deficiency from the Indian population.

Impairment of the phosphotransfer network and performance in broiler chickens experimentally infected by Eimeria spp.: The role of the oxidative stress.

The aim of this study was to evaluate whether infection Eimeria spp. in broiler chickens could negatively affect seric enzymes linked to adenosine triphosphate (ATP) metabolism and its relationship to oxidative stress. For this, 30 broiler chickens, 27 days-old, were divided into two groups (n = 15): the control group (C) and the group infected by Eimeria spp. (I). On days 1, 7 and 15 of the experiment, the animals were weighed, and fecal and blood samples were collected to evaluate the presence of oocysts and for serum biochemistry and enzymatic parameters, respectively. On day 15, one animal per repetition was submitted to euthanasia and intestinal fragments were collected for histopathological analyses. The body weight was lower in infected animals on day 15 of experiment, while oocyst counts were higher in infected animals on days 7 and 15 of the experiment. Serum levels of globulins were lower in infected animals on days 7 and 15 of experiment, while uric acid levels were higher in the same days, which represent changes on the immune system. Compared to the uninfected animals, on days 7 and 15, levels of serum globulins, triglycerides, creatine kinase and cholesterol were lower. Levels of adenylate kinase and reactive oxygen species (ROS) were higher on both days in infected animals, while levels of thiobarbituric acid-reactive substances (TBARS) were elevated on day 15. Lesions and immature forms of the parasite were observed in the intestines of infected birds. The phosphotransfer network elicited by an oxidative stress negatively affected the performance of broiler chickens with coccidiosis.

Changes in adiponectin, its receptors and AMPK activity in tissues of diet-induced diabetic mice.

AIM: A high-fat and high-sucrose diet (HFHSD) is usually used to induce type 2-like diabetes in animal models. We investigated the effect of HFHSD on serum and tissue levels of adiponectin, its receptors and AMP-activated protein kinase (AMPK) activity in adipose tissue, skeletal muscle and the liver. METHODS: C57Bl/6 male mice were fed either a standard diet or an HFHSD for four and 16 weeks, during which time glucose and insulin tolerance tests were performed. RESULTS: After four weeks, the HFHSD-fed mice were obese and glucose-intolerant and, after 16 weeks, they were obese and diabetic. In general, four weeks of HFHSD feeding did not modify either circulating or tissue adiponectin levels, nor adiponectin receptors or AMPK activity in the tissues studied. A significant increase of circulating adiponectin was observed after 16 weeks of HFHSD feeding, whereas adiponectin expression was decreased in adipose tissue. Muscle expression of adiponectin was increased at 16 weeks in terms of both mRNA and protein levels, and correlated to adipose-specific gene expression. However, AdipoR1 mRNA levels and AMPK activity were decreased in muscle at 16 weeks, suggesting decreased sensitivity to adiponectin in the muscle of diabetic mice. Finally, liver adiponectin expression was detectable only at protein levels and was increased in HFHSD mice at 16 weeks, suggesting "contamination" by circulating adiponectin. AdipoR2 mRNA levels were significantly decreased, whereas AMPK was increased, in the liver at 16 weeks. CONCLUSION: Overall, our data suggest that HFHSD-induced diabetes is not associated with adiponectin deficiency, but with tissue-specific defects of adiponectin-receptor expression and AMPK activity.

Erythrocyte adenylate kinase deficiency: characterization of recombinant mutant forms and relationship with nonspherocytic hemolytic anemia.

OBJECTIVE: Red cell adenylate kinase (AK) deficiency is a rare hereditary erythroenzymopathy associated with moderate to severe nonspherocytic hemolytic anemia and, in some cases, with mental retardation and psychomotor impairment. To date, diagnosis of AK deficiency depends upon demonstration of low enzyme activity in red blood cells and detection of mutations in AK1 gene. To investigate the molecular bases of the AK deficiency, we characterized five variants of AK1 isoenzyme-bearing mutations (118G>A, 190G>A, 382C>T, 418-420del, and 491A>G) found in AK-deficient patients with chronic hemolytic anemia. MATERIALS AND METHODS: The complete AK1 cDNA was obtained by standard procedures and using as template the reticulocyte RNA. The cDNA was cloned in a plasmid vector and the enzyme was expressed in Escherichia coli BL21(DE3)pLysS, and purified by standard protocols to homogeneity. DNA mutants bearing point mutations were obtained from the cloned wild-type cDNA using standard methods of site-directed mutagenesis, whereas the DNA mutant with deletion of codon 140 was obtained by a two-step method. RESULTS: Four mutant enzymes (Gly40Arg, Gly64Arg, Arg128Trp, Asp140del) were severely affected in activity, displaying a catalytic efficiency of four orders of magnitude lower than the wild-type; one (Tyr164Cys) was grossly perturbed in protein stability. CONCLUSIONS: The altered properties displayed by the mutant enzymes support the cause-effect relationship between AK1 mutations and hemolytic anemia.

Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

A child with hemolytic anemia was found to have severe erythrocyte adenylate kinase (AK) deficiency, but an equally enzyme-deficient sibling had no evidence of hemolysis. No residual enzyme activity was found in erythrocytes by spectrophotometric methods that could easily have detected 0.1% of normal activity. However, concentrated hemolysates were shown to have the capacity to generate small amounts of ATP and AMP from ADP after prolonged incubation. Hemolysates could also catalyze the transfer of labeled gamma-phosphate from ATP to ADP. Intact erythrocytes were able to transfer phosphate from the gamma-position of ATP to the beta-position, albeit at a rate substantially slower than normal. They could also incorporate 14C-labeled adenine into ADP and ATP. Thus, a small amount of residual AK-like activity representing about 1/2,000 of the activity normally present could be documented in the deficient erythrocytes. The residual activity was not inhibited by N-ethylmaleimide, which completely abolishes the activity of the normal AK1 isozyme of erythrocytes. The minute amount of residual activity in erythrocytes could represent a small amount of the AK2 isozyme, which has not been thought to be present in erythrocytes, or the activity of erythrocyte guanylate kinase with AMP substituting as substrate for GMP. Peripheral blood leukocytes, cultured skin fibroblasts, and transformed lymphoblasts from the deficient subject manifested about 17, 24, and 74%, respectively, of the activity of the concurrent controls. This residual activity is consistent with the existence of genetically independent AK isozyme, AK2, which is known to exist in these tissues. The cause of hemolysis in the proband was not identified. Possibilities include an unrelated enzyme deficiency or other erythrocyte enzyme defect and intraction of another unidentified defect with AK deficiency.

Adenylate kinase 5 autoimmunity in treatment refractory limbic encephalitis.

We report two men with limbic encephalitis (LE) refractory to corticosteroids, IVIg and plasma exchange. Both patients had serum/CSF antibodies that reacted with the cytoplasm of neurons. Probing of a hippocampal cDNA library resulted in the isolation of adenylate kinase 5 (AK5). Patients' antibodies, but not those of 111 controls, recognized AK5-expressing phage plaques. Human AK5-affinity purified antibodies reproduced the neuronal immunolabeling of patients' antibodies, and co-localized with a rabbit AK5 antibody, confirming that the brain autoantigen was AK5. Detection of antibodies to AK5 in LE patients carries a poor prognosis, and suggests the prompt use of aggressive immunosuppression.

Metformin restores the penile expression of nitric oxide synthase in high-fat-fed obese rats.

Obesity is a well-known risk factor for erectile dysfunction, which is associated with reduced penile nitric oxide synthase (NOS) expression. Recently it was reported that metformin activates AMP-activated protein kinase (AMPK), which increases the expression of neuronal (n) NOS and endothelial (e) NOS. Thus, to evaluate whether metformin restores NOS expression in penile tissue, we measured penile expression of nNOS and eNOS after 4 weeks of metformin treatment (300 mg/kg/d) in 5-month-old high-fat-fed obese (HFO) rats. HFO rats have increased fat accumulation in visceral areas and marked suppression of nNOS and eNOS expression in penile tissue. However, metformin treatment decreased visceral fat deposition and restored nNOS and eNOS expression in penile tissue. The levels of AMPK and phosphorylated AMPK were also decreased in HFO rats but were subsequently elevated by metformin treatment. These results suggest that expression of NOS was suppressed by the high-fat diet but restored by metformin treatment. The effect of metformin on the expression of NOS may be associated with its activation of AMPK.

An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further investigation.

Muscle adenylate kinase in Duchenne muscular dystrophy.

On the basis of electrophoretic and enzyme inhibition studies it was postulated that an aberrant adenylate kinase occurs in muscle and serum of patients with Duchenne muscular dystrophy (Schirmer, R.H. and Thuma, E. (1972) Biochim. Biophys. Acta 268, 92-97; Hamada, M. et al. (1981) Biochim. Biophys. Acta 660, 227-237; Hamada et al. (1985) J. Biol. Chem. 260, 11595-11602). On the basis of the following results we conclude that Duchenne muscular dystrophy patients do not possess an unusual adenylate kinase isoenzyme. In muscle biopsies from five Duchenne patients, the electrophoretic mobility of adenylate kinase and the inhibition of the enzyme by P1, P5-di(adenosine-5')pentaphosphate (Ap5A) was normal. Because of the high SH-group content of the extracts from Duchenne muscle, high concentrations of Ellman's reagent were needed to inhibit adenylate kinase activity in these samples. In Duchenne plasma the adenylate kinase activity was elevated. Like in muscle specimens, the DTNB inhibition curves were shifted to higher reagent concentrations; this was due to a high SH-group content of Duchenne plasma when compared with normal plasma. With respect to inhibition by Ap5A and electrophoretic mobility, Duchenne adenylate kinase in Duchenne plasma behaved like normal muscle adenylate kinase in normal plasma. It was noted that normal muscle adenylate kinase changes its electrophoretic behaviour when mixed with normal or Duchenne plasma. This finding had been considered previously as evidence for the presence of an aberrant adenylate kinase in Duchenne plasma.

Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions.

Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H-labeled and unlabeled nucleotides and adenosine, direct transfer of gamma-terminal phosphate from [gamma-32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate-specificity and competitive studies, we identified three purine-inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5'-nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP-generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40-70 micro mol/l. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.

Multiple sprint work : physiological responses, mechanisms of fatigue and the influence of aerobic fitness.

The activity patterns of many sports (e.g. badminton, basketball, soccer and squash) are intermittent in nature, consisting of repeated bouts of brief (

MAPRE1 as a plasma biomarker for early-stage colorectal cancer and adenomas.

Blood-based biomarkers for early detection of colorectal cancer could complement current approaches to colorectal cancer screening. We previously identified the APC-binding protein MAPRE1 as a potential colorectal cancer biomarker. Here, we undertook a case-control validation study to determine the performance of MAPRE1 in detecting early colorectal cancer and colon adenoma and to assess the potential relevance of additional biomarker candidates. We analyzed plasma samples from 60 patients with adenomas, 30 with early colorectal cancer, 30 with advanced colorectal cancer, and 60 healthy controls. MAPRE1 and a set of 21 proteins with potential biomarker utility were assayed using high-density antibody arrays, and carcinoembryonic antigen (CEA) was assayed using ELISA. The biologic significance of the candidate biomarkers was also assessed in colorectal cancer mouse models. Plasma MAPRE1 levels were significantly elevated in both patients with adenomas and patients with colorectal cancer compared with controls (P < 0.0001). MAPRE1 and CEA together yielded an area under the curve of 0.793 and a sensitivity of 0.400 at 95% specificity for differentiating early colorectal cancer from controls. Three other biomarkers (AK1, CLIC1, and SOD1) were significantly increased in both adenoma and early colorectal cancer patient plasma samples and in plasma from colorectal cancer mouse models at preclinical stages compared with controls. The combination of MAPRE1, CEA, and AK1 yielded sensitivities of 0.483 and 0.533 at 90% specificity and sensitivities of 0.350 and 0.467 at 95% specificity for differentiating adenoma and early colorectal cancer, respectively, from healthy controls. These findings suggest that MAPRE1 can contribute to the detection of early-stage colorectal cancer and adenomas together with other biomarkers.

Phenotyping of eight erythrocytic enzymes in one acrylamide gel.

A procedure has been developed to phenotype eight erythrocytic enzymes, phosphoglucomutase (PGM1), adenylate kinase (AK), 6-phosphogluconate dehydrogenase (6PGD), adenosine deaminase (ADA), glyoxalase (GLO), esterase-D (EsD), acid phosphatase (AcP), and glutamic pyruvate transaminase (GPT) in one acrylamide gel and also to detect the presence of common abnormal hemoglobins. The agar overlay technic has been eliminated. This simplified procedure renders the phenotyping of erythrocytic enzymes practical in paternity testing.

Acute leukemia with t(1;3)(p36;q21), evolution to t(1;3)(p36;q21), t(14;17)(q32;q21), and loss of red cell A and Le(b) antigens.

At transformation of refractory anemia with ring sideroblasts to acute nonlymphocytic leukemia (ANLL) the bone marrow cells of a 75-year-old woman showed three different karyotypes, i.e., 46,XX,46,XX,t(1;3)(p36;q21) and 46,XX,t(1;3)(p36;q21),t(14;17)(q32;q21). She received no antileukemic therapy, and 1 year later, all her bone marrow cells were t(1;3)(p36;q21),t(14;17)(q32;q21). In association with the onset and first 11 months of ANLL, the platelet count increased 10-fold to a peak of 750 x 10(9)/L, providing further evidence that the t(1;3)(p36;q21) translocation causes stimulation of thrombopoiesis. Six months after transformation, her red cells showed reduced expression of A and Leb antigens. Serum alpha-n-3-acetylgalactosaminyl transferase (blood group A transferase) and red cell adenylate kinase were both reduced. The genes for both these substances are at 9q34, which suggests an abnormality here, although cytogenetically chromosome 9 appeared normal. This is the first case with t(1;3)(p36;q21) to show concurrent loss of red cell antigens and the first report detailing the course of untreated ANLL with t(1;3)(p36;q21).

Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase.

A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions.

Carrier detection in X-linked recessive (Duchenne) muscular dystrophy: pyruvate kinase isoenzymes and creatine phosphokinase in serum and blood cells.

Allosterism allows individual assay of both isoenzymes, one abundant in muscle, of pyruvate kinase (PK), recently reported superior to serum creatine phosphokinase (CPK) in detecting patients with and female carriers of X-linked recessive (Duchenne) muscular dystrophy (DMD). Extensive comparative studies did not support these findings and confirmed the marked superiority of CPK over rariants of PK or other enzymes in sensitivity, stability and convenience. Deducting the adenylate kinase increment (AKI) further refined the CPK assay, eliminating the effect of haemolysis in diagnosis and enabling studies of blood cell content. Both leucocytes and erythrocytes liberated PK and lactate dehydrogenase (LDH) after brief chilling or disruption. Only erythrocytes showed a CPK content, however, constantly adjusted to match that of serum as if by free cell membrane passage, but less accomodating to a sudden large influx of CPK than of LDH, where an apparent buffering effect could account for differences in clinical response.