Cardiovascular adverse events associated with immune checkpoint inhibitors: A retrospective multicenter cohort study.

BACKGROUND: Over the past decade, immune checkpoint inhibitors (ICIs) have significantly transformed cancer treatment. However, ICIs inevitably may cause a spectrum of immune-related adverse events, among which cardiovascular toxicity, particularly myocarditis, while infrequent, has garnered increasing attention due to its high fatality rate. METHODS: We conducted a multicenter retrospective study to characterize ICI-associated cardiovascular adverse events. Logistic regression was performed to explore the risk factors for the development of myocarditis and severe myocarditis. Receiver operating characteristic curves were conducted to assess the diagnostic abilities of cardiac biomarkers to distinguish different cardiovascular toxicities, and the performance and calibration were evaluated using Hosmer-Lemeshow test. RESULTS: Forty-four patients were identified, including thirty-five myocarditis, five heart failure, three arrhythmias, and one myocardial infarction. Compared with other patients, myocarditis patients had higher cardiac troponin-I (cTnI) levels (p < 0.001), higher creatine kinase levels (p = 0.003), higher creatine kinase isoenzyme-MB (CK-MB) levels (p = 0.013), and shorter time to the incidence of adverse cardiovascular events (p = 0.022) after ICI treatment. Twenty-one patients (60%) were classified as severe myocarditis, and they presented higher cardiac troponin I (cTnI) levels (p = 0.013), higher N-terminal pro-B-type natriuretic peptide levels (p = 0.031), higher creatine kinase levels (p = 0.018), higher CK-MB levels (p = 0.026), and higher neutrophil to lymphocyte ratio (NLR) levels (p = 0.016) compared to non-severe myocarditis patients after ICI treatment. Multivariate logistic regression showed that CK-MB (adjusted odds ratio [OR]: 1.775, 95% confidence interval [CI]: 1.055-2.984, p = 0.031) was the independent risk factor of the development of ICI-associated myocarditis, and cTnI (adjusted OR: 1.021, 95% CI: 1.002-1.039, p = 0.03) and NLR (adjusted OR: 1.890, 95% CI: 1.026-3.483, p = 0.041) were the independent risk factors of ICI-associated severe myocarditis. The receiver operating characteristic curve showed an area under curve of 0.785 (95% CI: 0.642 to 0.928, p = 0.013) for CK-MB, 0.765 (95% CI: 0.601 to 0.929, p = 0.013) for cTnI, and 0.773 for NLR (95% CI: 0.597 to 0.948, p = 0.016). CONCLUSIONS: Elevated CK-MB after ICI treatment is the independent risk factor for the incidence of ICI-associated myocarditis, and elevated cTnI and NLR after ICI treatment are the independent risk factors for the development of ICI-associated severe myocarditis. CK-MB, cTnI, and NLR demonstrated a promising predictive utility for the identification of ICI-associated myocarditis and severe myocarditis.

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors.

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

[Screening for Duchenne muscular dystrophy in newborns in the Ningxia region]. / å®å¤åœ°åŒºæ–°ç”Ÿå„¿æœæ°è‚Œè¥å »ä¸è‰¯ç—‡ç­›æŸ¥.

OBJECTIVES: To evaluate the incidence rate of Duchenne muscular dystrophy (DMD) in the male newborns in the Ningxia region and establish a critical threshold for screening DMD in newborns to distinguish between the normal population and affected individuals. METHODS: A total of 10 000 male newborns were screened using immunofluorescence analysis of creatine kinase isoenzyme concentrations in heel spot dried blood specimens. Newborns with the concentrations higher than the critical threshold were recalled for serum creatine kinase measurements. Genetic testing was performed to confirm diagnosis in cases showing abnormalities. RESULTS: Among the screened 10 000 male newborns, two were confirmed to have DMD through genetic testing, resulting in a preliminary estimated incidence rate of 1/5 000 for male newborns in the Ningxia region. The critical threshold for creatine kinase isoenzyme concentration in newborns in this region was determined to be 468.57 ng/mL. CONCLUSIONS: Screening for DMD in newborns is feasible in the Ningxia region. Early screening, diagnosis, and treatment of DMD can improve the quality of life for affected individuals and help families make informed decisions regarding further pregnancies.

Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

Diagnostic Accuracy of Creatine Kinase Isoenzyme-MM Test in Newborn Screening for Duchenne Muscular Dystrophy: A Systematic Review and Meta-Analysis.

BACKGROUND: To systematically evaluate the diagnostic accuracy of the creatine kinase isoenzyme-MM (CK-MM) test in newborn screening for Duchenne muscular dystrophy (DMD). METHODS: A comprehensive literature search was conducted up to October 31, 2022, in PubMed, Embase, Cochrane Library, Web of Science, and Scopus Database. To evaluate the diagnostic value, the sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), area under the curve (AUC), and Q∗ index were pooled. Threshold effect followed by subgroup analysis and meta-regression were performed to explore the source of heterogeneity. Sensitivity analysis was used to verify the robustness of the findings. RESULTS: A total seven studies with 248,853 newborns was included in our meta-analysis. The pooled SEN and SPE were 1.00 (95% confidence interval [CI]: 0.89∼1.00) and 1.00 (95% CI: 1.00 to 1.00), respectively; the PLR and NLR were 1004.59 (95% CI: 251.37∼4014.91) and 0.13 (95% CI: 0.05∼0.34), respectively; the DOR was 877.96 (95% CI: 983.24∼78,366.32); the AUC and Q index were 0.8683 and 0.9326, respectively. Sensitivity analysis showed that two studies had an impact on the pooled results and mainly contributed to the heterogeneity. CONCLUSIONS: CK-MM test demonstrated high accuracy in newborn screening for DMD and may be a valuable alternative in the early diagnosis of the disease followed by confirmatory genetic testing.

Associations between antibiotic exposure and abnormal cardiac enzyme profiles in older Chinese adults.

Biomonitoring methods can be used to measure exposure to antibiotics in the general population; however, epidemiological data on the associations between urinary antibiotic levels and the cardiac profiles of enzymes lactate dehydrogenase, creatine kinase, and creatine kinase isoenzyme in older adults remain sparse. We investigated these associations in 990 individuals from the Cohort of Elderly Health and Environment Controllable Factors. Antibiotic residues in urine samples were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Urinary levels of 34 antibiotics were measured. The participants' cardiac enzyme profiles were influenced by sex, age, marital status, education level, cohabitation status, physical activity, dietary structure, body mass index, depression presence and salt, sugar, and oil consumption (P < 0.05). Oxytetracycline, tetracycline, doxycycline, sulfaclozine, and, florfenicol concentrations were negatively associated with the risk of having an abnormal cardiac enzyme profile. Older adults exposed to higher concentrations of norfloxacin had a higher risk of LDH anomalies. After antibiotics were classified, we identified associations between exposure to chloramphenicols, sulfonamides, or veterinary antibiotics and a lower risk of having an abnormal cardiac enzyme profile. Obtaining an accurate epidemiological profile of antibiotic exposure is indispensable for the prevention and detection of cardiac enzyme profile abnormalities in older adults.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Metabolic priming in G6PDH isoenzyme-replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning.

We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH-isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress-related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non-stressed plants contained twofold elevated fructose-2,6-bisphosphate (F2,6P2 ) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose-to-sucrose ratios, but also more lipids. Above-ground biomass lay in between susceptible Xanthi and resistant SNN, with photo-assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid-to-carbohydrate ratios, resulting in increased harvest yields - also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non-stressed plants. In leaves of salt-watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non-stressed plants showed enhanced PSII-induction kinetics (upon dark-light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids - especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi-cP2 lines. Apparently, metabolic priming by F2,6P2 -combined with sustained NADP(H) turnover-bypasses the genetically fixed growth-defence trade-off, rendering tobacco plants more stress-resilient and productive.

Creatine Kinase MB Isoenzyme Is a Complementary Biomarker in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an invariably fatal neurodegenerative disease with limited therapeutic options. There is an urgent need for novel biomarkers to be used as surrogates for new therapeutic trials and disease monitoring. In this study, we sought to systematically study creatine kinase isoenzyme MB (CK-MB) in a real-world cohort of ALS patients, assess the diagnostic performance, and evaluate its association with other laboratory and clinical parameters. We reviewed data from 194 consecutive patients that included 130 ALS patients and 64 disease control patients (primary lateral sclerosis [PLS], benign fasciculations syndrome [BFS], Huntington's disease [HD] and Alzheimer's disease [AD]). CK-MB was elevated in the sera of more than half of all patients with ALS. In patients with spinal-onset ALS, CK-MB levels were significantly higher than in patients with other neurodegenerative diseases. Patients with slower rates of functional decline had a significantly higher baseline CK-MB. Furthermore, CK-MB elevations correlated with cardiac troponin T (cTnT) and with revised ALS Functional Rating Scale (ALSFRS-R) bulbar subcategory. We posit that measuring CK-MB in ALS patients in a complimentary fashion could potentially aid in the diagnostic workup of ALS and help discriminate the disease from some ALS mimics and other neurodegenerative diseases. CK-MB levels also may provide valuable prognostic information regarding disease aggressiveness as well as correlations with specific phenotypic presentations.

A study of the interaction space of two lactate dehydrogenase isoforms (LDHA and LDHB) and some of their inhibitors using proteochemometrics modeling.

Lactate dehydrogenase (LDH) is a tetramer enzyme that converts pyruvate to lactate reversibly. This enzyme becomes important because it is associated with diseases such as cancers, heart disease, liver problems, and most importantly, corona disease. As a system-based method, proteochemometrics does not require knowledge of the protein's three-dimensional structure, but rather depends on the amino acid sequence and protein descriptors. Here, we applied this methodology to model a set of LDHA and LDHB isoenzyme inhibitors. To implement the proteochemetrics method, the camb package in the R Studio Server programming environment was used. The activity of 312 compounds of LDHA and LDHB isoenzyme inhibitors from the valid Binding DB database was retrieved. The proteochemometrics method was applied to three machine learning algorithms gradient amplification model, random forest, and support vector machine as regression methods to find the best model. Through the combination of different models into an ensemble (greedy and stacking optimization), we explored the possibility of improving the performance of models. For the RF best ensemble model of inhibitors of LDHA and LDHB isoenzymes, and were 0.66 and 0.62, respectively. LDH inhibitory activation is influenced by Morgan fingerprints and topological structure descriptors.

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells.

INTRODUCTION: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. OBJECTIVES: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. METHODS: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. RESULTS: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62. CONCLUSION: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

The effect of phytoprotectors on the functioning of liver NAD⁺- and NADP⁺-maliс enzymes in rats with alloxan diabetes.

The development of experimental alloxan diabetes in rats was accompanied by the increase the activity of liver NAD⁺- and NADP⁺-dependent malic enzymes (ME; NAD⁺-ME, EC 1.1.1.39 and NADP⁺-ME, 1.1.1.40) associated with an increase in the rate of transcription of genes encoding these enzymes. Oral administration of aqueous extracts of Jerusalem artichoke and olive to diabetic rats caused a noticeable decrease in blood glucose, a decrease in the rate of transcription of the studied genes; and a decrease in ME activity towards normal values. Thus, extracts of Jerusalem artichoke and olive can be used as additives to the standard therapy of diabetes mellitus.

Synergism and toxicity of iron nanoparticles derived from Trigonella foenum-graecum against pyrethriod treatment in S. litura and H. armigera (Lepidoptera: Noctuidae).

The tobacco cutworm, Spodoptera litura and cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) are important pests of various agricultural crops that cause sevier economic loses throughout the world. Indiscriminate and frequent use of insecticide may lead to development of resistance in these pests. Nanotechnology has given an alternative to manage and overcome insecticide resistance for pest management strategies. In the present study the iron nanoparticles derived from Trigonella foenum-graecum leaf extract (FeNPs) was investigated for its ecofriendly management of pyrethroid resistance in two lepidopteron pest species at 24 h, 48 h and 72 h post treatment. The result showed high mortality (92.83% and 91.41%) of S. litura and H. armigera at 72 h treatment upon FeNPs and fenvalerate (Fen + FeNPs) teratment. Probit analysis revealed high LC50 upon Fen + FeNPs treatment (130.31 and 89.32 mg/L) with a synergism ratio of 1.38 and 1.36. Antifeedant activity of six dofferent concentration of FeNPs revelaed increased antifeedant activity with respect to increasing concentration of nanoparticles ranging from 10 to 90% and 20-95% againt both insects (p<0.05). Detoxification activity of carboxylesterase was elevated at 630 µmol/mg protein/min (p<0.05) in fenvalerate treatment, whereas decreased activity was found (392umole/mg protein/min) in FeNPs and Fen + FeNPs treatment (P<0.001). GST and P450 activity was also increased in fenvalerate treatment, whereas decreased activity was observed in FeNPs and Fen + FeNPs. Esterase isoenzyme banding pattern revealed four bands in fenvalerate treatment and two bans (E3 and E4) in Fen + FeNPs combination. Hence the present study concludes that T. foenum-graecum synthesized iron nanoparticles could be an effective alternate for ecofriendly management of S. litura and H. armigera.

Expression Levels of Glutathione S-Transferase and Cytochrome P450 Isoenzymes in Nasal Polyp Tissue.

Nasal polyps are benign sinonasal masses composed of eosinophils and extracellular edema. Pathogenesis of the polyp formation is unclear but several studies strongly suggest a correlation with infection, inflammation and allergy conditions. Our aim is to investigate the potential link between allergy and nasal polyp in tissue level. Nasal polyp group included 60 patients whose diagnosis was confirmed with biopsy and the control group included 38 healthy patients. Tissue sample of the control group was taken from inferior turbinate mucosa under local anesthesia and nasal polyp tissue was collected from functional endoscopic sinus surgery. The glutathione S-transferase (GST) and cytochrome P450 (CYP) isoenzyme expressions of the tissue samples were investigated under light microscopy and graded by a senior pathologist. GSTP1 protein expression was significantly higher in tissue samples from nasal polyp group compared to that of control group (p < 0.05). However, CYP1A1, GSTM1 and GSTA1 isoenzymes were not different between the two groups (p > 0.05). We have found that GSTP1 isoenzyme was elevated in nasal polyp tissue compared to the control. The increase in protein expression of GSTP1 might have occured as a tissue response to the increased oxidative stress thus suggesting a role of GSTP1 in polyp formation.

Effect of geographical factors on reference values of creatine kinase isoenzyme.

The aim of this study was to investigate the geographical spatial distribution of creatine kinase isoenzyme (CK-MB) in order to provide a scientific basis for clinical examination. The reference values of CK-MB of 8697 healthy adults in 137 cities in China were collected by reading a large number of literates. Moran index was used to determine the spatial relationship, and 24 factors were selected, which belonged to terrain, climate, and soil indexes. Correlation analysis was conducted between CK-MB and geographical factors to determine significance, and 9 significance factors were extracted. Based on R language to evaluate the degree of multicollinearity of the model, CK-MB Ridge model, Lasso model, and PCA model were established, through calculating the relative error to choose the best model PCA, testing the normality of the predicted values, and choosing the disjunctive kriging interpolation to make the geographical distribution. The results show that CK-MB reference values of healthy adults were generally correlated with latitude, annual sunshine duration, annual mean relative humidity, annual precipitation amount, and annual range of air temperature and significantly correlated with annual mean air temperature, topsoil gravel content, topsoil cation exchange capacity in clay, and topsoil cation exchange capacity in silt. The geospatial distribution map shows that on the whole, it is higher in the north and lower in the south, and gradually increases from the southeast coastal area to the northwest inland area. If the geographical factors are obtained in a location, the CK-MB model can be used to predict the CK-MB of healthy adults in the region, which provides a reference for us to consider regional differences in clinical diagnosis.

Agarose gel electrophoresis pattern of serum creatine kinase and lactate dehydrogenase isoenzymes in zoo-managed Asian elephants (Elephas maximus).

Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes were evaluated in nine zoo-managed Asian elephants (Elephas maximus) using a commercial agarose gel electrophoresis (AGE) kit. CK was separated into two major fractions, CK-BB and CK-MM, along with a small fraction of macroenzyme-CK type 2 (mCK2); CK-MM was the largest fraction. LDH was separated into five fractions (LDH1-5); LDH3 was the largest fraction. Age was negatively and positively correlated with the percentages of CK-BB and CK-MM, respectively, and negatively correlated with CK-BB and mCK2 activities. These results indicate that an AGE kit can be used to evaluate CK and LDH isoenzymes. Routine isoenzyme testing may enable early detection of disease and physiological changes.

Practical Considerations for the Clinical Application of Bone Turnover Markers in Osteoporosis.

Bone turnover markers (BTMs) are released during the bone remodelling cycle and are measurable in blood or urine, reflecting bone remodelling rate. They have been useful in elucidating the pharmacodynamics and effectiveness of osteoporosis medication in clinical trials and are increasingly used in routine clinical management of osteoporosis, especially for monitoring therapy, in addition to their use in other metabolic bone disease such as Paget's disease of bone and osteomalacia. Serum ß isomerised C-terminal telopeptide of type I collagen and pro-collagen I N-terminal propeptide have been designated as reference BTMs for use in osteoporosis. In addition, bone-specific isoenzyme of alkaline phosphatase (B-ALP) secreted by osteoblasts and tartrate-resistant acid phosphatase 5b (TRACP-5b) secreted by osteoclasts are also found to be specific markers of bone formation and resorption, respectively. The concentrations of the latter enzymes in blood measured by immunoassay provide reliable measures of bone turnover even in the presence of renal failure. B-ALP is recommended for use in the assessment of renal bone disease of chronic kidney disease, and TRACP-5b shows promise as a marker of bone resorption in that condition. BTMs in blood do not suffer from biological variation to the same extent as the older BTMs that were measured in urine. Appropriate patient preparation and sample handling are important in obtaining accurate measures of BTMs for clinical use. Reference change values and treatment targets have been determined for the reference BTMs for their use in monitoring osteoporosis treatment. Further ongoing studies will enhance their clinical applications.

Quality and Purity of L929 Cell Cultures: Isoenzyme Profiles As a Sensitive Marker of Cross-Contamination.

Objective: Inappropriate handling of cells can generate modifications in the genomic DNA. The additional risk is cross-contamination. Isoenzyme analysis with gel agarose electrophoresis is a known, fast, and cheap technique for detection of species-specific isoforms of intracellular enzymes. The aim of the experimental work was to check if variations in the cell growth conditions can affect morphology and/or nuclear anomalies including micronuclei (MN) in the L929 cells; and to define how sensitive and selective is the classic gel agarose electrophoresis for analysis of isoforms of the selected enzymes to detect cross-contamination of L929 cultures with HeLa cells or with the related species, such as CHO-K1 cells, in the case of unavailability of the commercial kits. Methods: The experiments were done with use of the National Collection of Type Cultures clone 929 (L929)-mouse fibroblasts from subcutaneous connective tissue; HeLa-human cervix adenocarcinoma; and CHO-K1-epithelial-like hamster ovary cells. Cell morphology was evaluated with a light/fluorescence microscope. MN were determined with the cytokinesis-block micronucleus assay, and the isoenzyme analysis was performed using gel agarose electrophoresis. Results: As shown, the overgrown cultures result in a significant increase of the MN in the L929 cells. The band patterns for lactate dehydrogenate, glucose-6-phosphate dehydrogenase, or malate dehydrogenase allow identification of the single L929, HeLa, or CHO-K1 cell line and to detect the cross-contamination, even up to 0.4%. Conclusions: There can be no exceptions from the recommended cell culture conditions in the passage scheme. The sensitivity of the gel agarose separation depends on the cells and on the type of enzyme tested and seems to be sufficient in a quick screening of the CHO-K1, L929, or HeLa cell cultures through the possible mutual contamination.

The serum activities of alkaline phosphatase isoenzymes measured using two approved methods in zoo-managed Asian elephants (Elephas maximus).

The approved Japanese measurement method of circulating alkaline phosphatase (ALP) has changed from that of the Japan Society of Clinical Chemistry (JSCC) to that of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). We measured the serum levels of total ALP (t-ALP) and those of the isoenzymes ALP2 and ALP3 in 50 Asian elephant (Elephas maximus) specimens using both methods. The activities determined by the IFCC method were roughly one-third lower than those determined by the JSCC method. We present conversion formulae. Our results enable comparisons of historical and current data on serum ALP activities in endangered, zoo-managed Asian elephants.

Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris.

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.