Isoenzyme Distribution Research Articles

Comparative Analysis of Catalase Activity in Plants: Spectrophotometry and Native PAGE Approaches.

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme.

Creatine kinase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines.

Creatine kinase (CK) exists as three isoenzymes (CK-MM, CK-MB, and CK-BB) that are predominantly expressed in specific tissues and can be detected in both the serum and cerebrospinal fluid (CSF). CSF CK has been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total CSF CK activity can indicate neurologic abnormalities. The purpose of this study was to establish reference intervals for CK and its three major isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive reference interval for this enzyme in healthy canines, the diagnostic use and possible significance of CK in clinical disease can be studied. Serum and/or CSF were collected from healthy dogs. Total CK activity was measured spectrophotometrically, and isoenzyme distributions were determined using the QuickGel CK Vis Isoenzyme Kit and a densitometric scanner. Total CK and CK isoenzyme activities were determined within 8 h of collection. The median serum total CK in healthy canines was 159.0 U/L (range: 53.0-539.0 U/L), while the median CSF total CK was 3.7 U/L (range: 2.0-84.0 U/L). CK-BB and CK-MM were approximately equal in the serum, while CK-MM was the predominant isoenzyme in the CSF. Knowledge of the normal distribution and concentration of CK in canine serum and CSF will set the foundation for future studies of canine CK as a potentially clinically useful biomarker.

Study of variability alpha-amylase isozymes in wheat varieties of Ukrainian breeding

Aim. To investigate the variability of alpha-amylase isoenzymes in soft wheat varieties of Ukrainian selection, created by breeding institutions located in different climatic zones (Forest Steppe and Steppe). Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the «Falling Number 1700» was determined. Results. For a representative assessment of the distribution of alpha-amylase isoenzymes, we studied the zoned varieties of common wheat from two breeding centers located in different climatic zones: the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kyiv (Forest-steppe) and the Breeding and Genetics Institute of the National Academy of Sciences, Odessa (Steppe). The variability of wheat grain alpha-amylase was revealed. A search for associations between alpha-amylase variants and resistance to pre-harvest grain germination was conducted. Conclusions. The prevalence of alpha-amylase isoenzyme variants in Ukrainian common wheat varieties was estimated. Among Odessa varieties, the AbCd alpha-amylase variant predominates with a frequency of 50.0 %. AbCd and AbCD phenotypes with a frequency of 38.8 % predominate in the selection varieties of the IFRG of the National Academy of Sciences of Ukraine. It was shown that the genotypes containing the AbCD and ABсD isoenzyme variant are more resistant to pre-harvest germination, and the ABCd and AbcD variants are less resistant to pre-harvest grain germination.

The impaired distribution of adenosine deaminase isoenzymes in multiple sclerosis plasma and cerebrospinal fluid.

BackgroundAdenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor.AimsThis study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes.Methods and resultsThe enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients.ConclusionIn this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.

Abstract P5-17-01: Targeting Acetyl-CoA carboxylase in pre-clinical breast cancer models

Abstract Background: Cancer cells and normal cells of the same lineage differ in their metabolism. We previously described large scale shifts in isoenzyme distribution between matching cancer and normal tissues and identified Acetyl-coA carboxylase (ACC1/ACACA) as a cancer dominant enzyme that is overexpressed in multiple cancer types. ACC1 catalyzes the initial rate-limiting step in de novo fatty acid synthesis, the conversion of acetyl-CoA to malonyl-CoA. Gene knock-out experiments demonstrated that this enzyme is essential for cancer growth. In this study, we evaluated the in vitro and in vivo efficacy of a small molecule ACC inhibitor, PF05175157 as a potential anticancer drug. This drug has been tested in clinical trials for diabetes, but development was discontinued due to grade 2 thrombocytopenia.Methods: We performed in vitro cytotoxicity assays in 15 breast cancer cell lines and in normal mammary epithelial HMEC cells, examined effect on apoptosis and cell cycle progression, and tested for synergy with alpelisib, docetaxel, doxorubicin, everolimus, iniparib, neratinib and TEPP46 (PKM2 and PKLR activator). We next assessed in vivo single agent activity in a triple negative patient derived (PDX) model (J000102184) in NSGTM mice and in MDAMB468 xenografts implanted into Rag2/IL2RG double knockout mice. We performed RNA sequencing and metabolomic profiling of cells treated with PF05175157 to study metabolic and transcriptomic effects of the drug. Results: PF05175157 induced time and dose dependent growth inhibition in all but 1 of the 15 cancer cell lines. The estimated EC50 after 72h exposure ranged from 0.95 to 76 μg/mL in T47D and BT549 cells, respectively (Cmax of 20 μg/mL can be achieved in human serum). There was no significant inhibitory effect on HMEC cells. In cancer cell lines, the % of apoptotic cells increased from 4% to 8% in BT474 and from 7.7% to 17.8% in MDMBA468 cells upon treatment with the compound, and there was a trend towards G2/M cell cycle arrest in both cell lines after 72 hours of exposure (10μg/mL). In drug combination experiments, PF05175157 added to iniparib, or to the PKM2 activator, TEPP46, decreased cell viability compared to single agent therapies in several cell lines. PF05175157 significantly delayed tumor growth compared to vehicle, when administered orally (20mg/kg gavage BID) in a TNBC PDX model (median tumor volume after 33 days: 334.1 mm3 in PF05175157-treated vs. 490.5 mm3 in methylcellulose-treated mice; p=3.39e-7) and intraperitoneally (20 mg/kg in DMSO) in an MDAMB468 xenograft model (median tumor volume after 40 days: 244.5 mm3 in PF05175157-treated vs. 303.3 mm3 in DMSO-treated mice; p<0.05). Transcriptomic and metabolic profiling of MDAMB468 and BT474 cells treated with 10 ug/ml PF05175157 for 6 and 24 hours revealed activation of immune signaling, epigenetic regulation and DNA damage repair pathways along with down-regulation of a broad range of metabolic pathways. Conclusions: The small molecule ACC inhibitor, PF05175157, has significant single agent in vitro and in vivo growth inhibitory effect on a range of breast cancer cell lines at concentrations that can be achieved in human serum. It showed synergy with iniparib and another metabolic inhibitor (TEPP46). Targeting de novo fatty acid synthesis by inhibiting ACC is a promising therapeutic strategy. Citation Format: Julia Foldi, Michal Marczyk, Vignesh Gunasekharan, Tao Qing, Raghav Sehgal, Naing Lin Shan, Viswanathan Muthusamy, Sheila Umlau, Yulia V. Surovtseva, Richard Kibbey, Lajos Pusztai. Targeting Acetyl-CoA carboxylase in pre-clinical breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-01.

Isonzyme Changes and DNA Damage Associated with Sterility by Gamma Radiation in Trogoderma granarium Everts Males

The present study aimed to investigate the impact of gamma radiation on isoenzymes (peroxidase and polyphenyl oxidase) and DNA damage in Trogoderma granarium. The newly emerged male adults were irradiated with substerilizing and sterilizing doses of gamma radiation for the experiments (100 and 200Gy). The results showed that there was no difference in the distribution of peroxidase and polyphenyl oxidase isoenzymes, and the intensity of peroxidase isoenzymes was the same between the tested samples when compared to the control population and each other. There was a variation in the intensity of the detected polyphenyl oxidase isoenzyme. The DNA electrophoresis showed fragmentation in the gamma irradiated adults as compared to the control. Therefore, it could be concluded that these parameters could be used as a biomarker for identification of gamma irradiated T. granarium males.

Tamoxifen prevention of meningioma and its proposal for the treatment of meningioma. Revisiting old data in the light of recent epidemiological observations.

Epidemiological studies reported in 2016 and 2019 demonstrated that breast cancer patients under tamoxifen treatment had significantly reduced risks of meningioma development. Tamoxifen treatment duration longer than 1500 days or with cumulative dosage higher than 26 320 mg have especially lowered risk of meningioma. Clinical long-term anticancer efficacy of tamoxifen shall associate with simultaneous suppression of estrogen receptor and downregulation of certain growth factor pathways, which may associate with - but not limited to - protein kinase-C (PKC) signaling. In this study, we will put the evidence together and indicate that tamoxifen may be effective in meningioma treatment in some patients who do not express estrogen receptor but expresses PKC, yet much higher doses of tamoxifen will be needed to treat meningiomas than those applied to treat breast cancer. We underline the fact that immunohistochemical analysis of both estrogen receptor and PKC (especially α, δ, λ and ι isoenzymes) may guide in patient stratification for selective benefitting from tamoxifen in management of meningiomas. Lastly, it would also be logical to test individual responses of meningiomas to tamoxifen in primary monolayer and spheroid cultures before starting treatment in each patient as the differential distribution of PCK isoenzymes may cause also untoward effects.

Evaluation of total LDH and its isoenzymes as markers in preeclampsia.

Preeclampsia, a rapidly progressing pregnancy-specific multi-systemic syndrome is globally the leading cause of maternal and neonatal morbidity and mortality. This study aims to evaluate the serum total Lactate dehydrogenase levels in women with preeclampsia when compared to normotensive pregnant women and assess the electrophoretic pattern of the LDH isoenzymes in normal pregnancy, preeclampsia and eclampsia. The study, carried out in the Department of Biochemistry of MVJ Medical College, included 30 patients of preeclampsia and 30 normotensive gestational age-matched pregnant women admitted to the Department of OBG. Serum total LDH was analysed by DGKC method. Serum and cord blood samples for isoenzyme distribution analysis were collected from a normal pregnant woman undergoing delivery, a woman with mild eclampsia, two women with eclampsia, and analysed by slab gel electrophoresis followed by activity staining. LDH was significantly elevated in cases as well as between the case (mild and severe) groups, showed a moderate positive statistically significant correlation with systolic, diastolic blood pressure and a sensitivity of 50% and a specificity of 80%. Further, the isoenzyme pattern showed a decreasing distribution of aerobic forms of LDH in preeclampsia-eclampsia. Serum total LDH may serve as a robust and affordable marker of preeclampsia. Serum total LDH, along with its isoenzyme profile, might serve as a predictor and a stronger marker of preeclampsia when compared to serum LDH analysis alone. It may also be used to assess the severity of preeclampsia and hence help in predicting and preventing adverse maternal and foetal outcomes.

Reference intervals for the activity of lactate dehydrogenase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines

Lactate dehydrogenase (LD) exists as 5 isoenzymes (LD-1 through LD-5) that are expressed throughout the body and can be detected in both serum and cerebrospinal fluid (CSF). LD and its isoenzymes have been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total LD activity and atypical isoenzyme patterns can indicate disease processes, including neurologic abnormalities. The purpose of this study was to establish RIs for LD and its isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive RI for this enzyme in healthy canines, further study of the clinical and diagnostic usefulness of LD can be undertaken. Serum and atlantoaxial CSF were collected from clinically healthy dogs. Total LD activity was measured spectrophotometrically immediately after collection. Isoenzyme distributions were also determined within 8hours of collection using the QuickGel LD Isoenzyme technique and a densitometric scanner. The median serum total LD in healthy canines was 69.0 U/L (n=41; range: 21.0-217.0U/L), while the median CSF total LD was 10.0 U/L (n=40; range: 6.0-19.3U/L). LD-5 is the predominant isoenzyme in canine serum (n=40), contributing over half of the total enzyme activity. Conversely, in canine CSF (n=42), LD-1 is the predominant isoenzyme, followed by LD-2 and LD-3. Knowledge of the distribution and concentration of LD in the serum and CSF of healthy dogs will set the foundation for future studies of canine LD as a potentially clinically useful biomarker.

Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD+ . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc.

An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Male Urethra

To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.

Infection of sheep with Strongyloides papillosus: The effect of intensity of parasitic infection as well as therapy with albendazole on certain biochemical parameters in the blood of sheep

The objective of this work was to investigate certain biochemical parameters in the blood of sheep in conditions of different intensity of parasitic infection with Strongyloides papillosus, and also after the therapy with albendazole. The research was carried out on the sheep of Wurtemberg breed (n = 30) in which there was determined low, medium and high intensity of parasitic infection with S. papillosus. The control group (n = 10) consisted of the sheep that were negative to the presence of the parasitic infection. The degree and type of changes were monitored by determining the concentration of glucose, total protein, albumin, Albumin/Globulin ratio, AST, urea, total bilirubin, calcium, phosphorus, lactate dehydrogenase (LDH) total activity and isoenzyme distribution LDH1-5. On the basis of the obtained results it was concluded, through isoenzyme distribution of lactate dehydrogenase, that parasitic infection with S. papillosus can cause damage to the liver, heart muscle and lungs, while after the therapy with albendazole, the most affected organ is the liver. The concentration of glucose, total protein and albumin linearly decreased with the increase of intensity of the parasitic infection (p &lt; 0,05), and after the therapy with albendazole, a drop in concentration of these parameters was at a statistically significantly higher level (p&lt; 0,01). AST activity as well as the concentration of urea and total bilirubin grew linearly along with the intensity of the parasitic infection (p &lt; 0,05). After the therapy with ABZ, AST activity and concentration of total bilirubin were at a statistically significantly higher level (p &lt; 0,001), while urea concentration kept the same level as in the case of the parasitic infection. The values of calcium concentration (p &gt; 0,05) as well as phosphorus (p &lt; 0,05) linearly decreased with the parasitic infection intensity growth. Trend towards the decline of the concentration of these macroelements continues even after the therapy with albendazole (p &lt; 0,001). Considering our previous research in the field of oxidative stress, a phenomenon that is fundamentally related to the changes determined in this work, we strongly recommend that besides antihelmintics, preparations with antioxidative properties should be used in antiparasitic protocols as well. &lt;br&gt;&lt;font color="red"&gt;&lt;b&gt; This article has been retracted. Link to the retraction &lt;u&gt;&lt;a href="http://dx.doi.org/10.2298/VETGL190125001E"&gt;10.2298/VETGL190125001E&lt;/a&gt;&lt;u&gt;&lt;/b&gt;&lt;/font&gt;

Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes

Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

Determination of Oxidative Energy Metabolism and Plasma LDH Isoenzyme Patterns of Dolphins

In this study, oxidative energy metabolism was determined in 5 captive bottlenose dolphins (Tursiops truncatus), and subsequently compared to 6 Thoroughbred riding horses, and 12 lactating Holstein cows in order to assess symmorphosis of each animal species. Plasma metabolites profile, plasma MDH (M) and LDH (L) activities, M/L ratio, and plasma LDH isoenzyme distribution patterns were all examined. Overall, dolphins appear to have the greatest level of oxidative energy metabolism amongst horses and cows, due to having the greatest levels of plasma MDH activity. In addition, dolphin energy production/usage efficiency, via oxidative metabolism, was considered to be second (M/L ratio = 0.49) behind that of horses (M/L ratio = 0.79), possibly due to an increased reliance of non-oxidative energy metabolism over horses. In spite of demonstrating the lowest oxidative energy production/usage efficiency (M/L ratio = 0.15), cows also demonstrated the highest plasma LDH activity amongst all animal species. Although all animal groups displayed differing plasma LDH isoenzyme distribution patterns, dolphins and horses demonstrated a similarity with LDH-3 isoenzyme predominating in plasma; whereas LDH-1 isoenzyme predominates in cow plasma, thus inferring differences in oxidative/non-oxidative metabolism for energy generation/usage. Therefore, plasma MDH and LDH activity levels, M/L ratio, and plasma LDH isoenzyme pattern can all be useful indicators for better understanding oxidative energy metabolism and monitoring of captive animals' health. As it is not easy to obtain tissue samples from animals, the development of blood indicators for evaluating whole body metabolic state is necessary.

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation

BackgroundAlkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches.MethodsMice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects.ResultsAP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS.ConclusionsOur findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

Effects of infection intensity with Strongyloides papillosus and albendazole treatment on development of oxidative/nitrosative stress in sheep

The objective of this study was to estimate and evaluate oxidative/nitrosative stress parameters in sheep infected with Strongyloides papillosus and after antihelminthic treatment with albendazole (ABZ). This parasite, especially during development stages can seriously damage parenchaematous organs during migration within the host. The presence of parasites leads to increased productions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is also well known that certain drugs can be very harmful for the delicate oxidant–antioxidant equilibrium, provoking oxidative stress during their biotransformation. ABZ is a broad spectrum antihelminthic drug, frequently used in veterinary medicine for therapy of parasitic infections. The current research was performed on female Württemberg sheep (n=48). The distribution of parasites in sheep was evaluated using the native smear coprological technique, by sedimentation and flotation methods, revealing the presence of S. papillosus. The degree of infection intensity per sheep was quantitatively established by the method of McMaster, the animals having been divided into three groups according to the intensity of infection; mild, moderate and high. The control group consisted of sheep negative to the parasites. After determining the type of parasite infection, the sheep were treated with ABZ, per orally, in single doses of 5mg/kg per body weight. Sampling of feces for parasitological and blood for biochemical assaying was performed on the 0 and 21st day after treatment with ABZ. The oxidative stress parameters were measured for catalase activity (CAT), the red cell membrane damage by level of malondialdehyde (MDA), while carbonyl and thiol plasma protein group concentrations were used as indicators of the degree of protein oxidative modification. The activity of Cu,Zn-superoxide dismutase (SOD) and relative distribution of lactate dehydrogenase (LDH1–LDH5) activity were determined electrophoretically. The distribution of LDH isoenzymes in sheep moderately and highly infected with S. papillosus revealed that the parasite induced damage to the myocardial (LDH2), lung (LDH3) and liver cells (LDH5) in infected animals, while ABZ treatment only damaged liver cells (LDH5). The MDA concentration revealed that lipid peroxidation increased both in the presence of parasites and the antihelminthic formulation tested (p<0.001) when compared to the control sheep, while the increase of carbonyl concentration (p<0.001), as well as the observed decrease of thiol concentration (p<0.001) indicated significant oxidative damage of plasma proteins in experimental sheep, when compared to the control animals. Our results indicate that S. papillosus induces oxidative/nitrosative stress in sheep. The antihelminthic treatment with ABZ further promotes the disbalance of oxidative–antioxidative equilibrium in all tested sheep.

Matrix metalloproteinase-8/collagenase-2 in childhood otitis media with effusion

Background. Matrix metalloproteinases (MMPs), gelatinases, have been associated with otitis media with effusion (OME), but the role of collagenase-2/matrix metalloproteinase-8 (MMP-8) in OME has not been studied previously. We studied the levels, isoenzyme distribution, and activation of MMP-8 in childhood OME, and also the levels of pro- and active forms of MMP-2 and -9 as well as 120 kDa gelatinase complexes were assessed.Methods. Seventy middle ear fluid (MEF) samples were collected from 54 children with OME and classified to mucoid (n = 39) or serous (n = 31). MMPs were studied from MEF samples by time-resolved immunofluorometric assay, Western immunoblotting, and gelatin-zymography.Results. MMP-8 was found in its active form in MEF of children with OME. MMP-8 levels were significantly higher in mucous relative to serous OME. The pro- or active MMP-2 and -9 were found in MEF, but no MEF type-specific differences were found.Conclusion. Our results suggest that MMP-8 may play a role in the pathogenesis of childhood OME. New therapeutic strategies with MMP inhibitors targeting MMP-8, but allowing MMP-8 to carry out the protective action, may play a role in the future treatment of otitis media and OME. However, further studies of this topic are needed.

Associations Between Matrix Metalloproteinase‐8 and ‐14 and Myeloperoxidase in Gingival Crevicular Fluid From Subjects With Progressive Chronic Periodontitis: A Longitudinal Study

Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy. In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses. High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05). We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes.

Pattern of Cerebrospinal Fluid Lactic Dehydrogenase During Treatment of Bacterial Meningitis

Bacterial and aseptic meningitis are characterized by distinctive lactic dehydrogenase isoenzyme patterns. No studies have quantified the dynamics of lactic dehydrogenase isoenzyme distribution during treated bacterial meningitis. We used a retrospective case-series design, and reviewed files of all neonates with bacterial meningitis who attended our pediatric tertiary medical center for 8 years period. We identified neonates in whom a repeated lumbar puncture was indicated. Findings of cerebrospinal fluid analysis, including levels of lactic dehydrogenase isoenzymes, were compared with an age-matched reference group. In two patients with meningitis, lumbar puncture with cerebrospinal fluid analysis was repeated because of inadequate response to treatment or initially obscure etiologic pathogens. Both patients had initially low levels of lactic dehydrogenase-1 and lactic dehydrogenase-2 and high levels of lactic dehydrogenase-4 and lactic dehydrogenase-5, similar to other patients with bacterial meningitis. The distribution pattern of lactic dehydrogenase isoenzyme normalized after adequate antibiotic treatment. In light of the encouraging results in these two patients, further studies are warranted regarding the value of lactic dehydrogenase isoenzyme measurements for follow-up purposes and for evaluations of response to treatment.