Lactate Dehydrogenase Enzyme Activity Research Articles

Lactate dehydrogenase: An important molecule involved in acetamizuril action against Eimeria tenella

Lactate dehydrogenase (LDH), a vital enzyme in anaerobic glycolysis, is closely associated with the survival of parasites. Previous studies of some parasites have shown that LDH exhibits unique physicochemical properties and molecular structures and may be an ideal potential target for diagnosis and drug screening. In this study, we aimed to investigate the effects of acetamizuril, a novel anticoccidial compound, on LDH in the second-generation merozoites of Eimeria tenella (mz-LDH). Quantitative real-time PCR, Western blot, immunofluorescence and enzyme activity assays were each applied to detect the changes of mz-LDH. Our results indicated that the mRNA and protein levels of mz-LDH were reduced upon acetamizuril treatment. Immunolocalization of mz-LDH demonstrated that considerable amount of mz-LDH was distributed around or in the nuclei of second-generation merozoites within the untreated group; in contrast, the acetamizuril-treated group had very low level of mz-LDH. Meanwhile, LDH enzyme activity assay suggested that a decreased LDH enzyme activity in both cytoplasm and nucleus of merozoites in the acetamizuril-treated group. Moreover, the induced apoptosis in second-generation merozoites by the acetamizuril was evaluated by detecting caspase 3 activity, and acetamizuril was found to significantly increase caspase 3 activity. The above findings show that LDH may play an important role in the mediating the activity of acetamizuril against coccidiosis, and further investigation into this aspect might contribute to new light on the pathogenesis of E. tenella during its interaction with acetamizuril.

Cardioprotection of cortistatin against isoproterenol-induced myocardial injury in rats.

BackgroundThe present study was designed to examine whether cortistatin (CORT) could protect rats from myocardial injury induced by subcutaneously injecting isoproterenol (ISO) and to clarify the possible mechanisms.MethodsMale Sprague-Dawley (SD) rats were placed at random into four groups: the control group, the ISO group, the ISO + CORT 25 µg/(kg·d) group, and the ISO + CORT 50 µg/(kg·d) group. Rat models of myocardial injury were established with the subcutaneous (s.c.) injections of 85 mg/kg ISO for 2 days. In the ISO+ CORT 25 µg/(kg·d) group and ISO+ CORT 50 µg/(kg·d) group, rats were given s.c. injections of CORT 25 µg/(kg·d) and CORT 50 µg/(kg·d) on the day before ISO, 3 days, respectively. Serum malondialdehyde (MDA) content, lactate dehydrogenase (LDH) activity, and creatine kinase isoenzyme (CK-MB) activity were measured by corresponding test kits. Western blot was applied to evaluate the expression of endoplasmic reticulum stress-related protein glucose regulatory protein 78 (GRP78), enhancer-binding protein homologous protein (CHOP), cysteinyl aspartate specific proteinase-12 (caspase-12), LC3-II, Beclin-1, and p62 in the rat myocardium.ResultsCORT alleviated the increased enzyme activities of serum LDH and CK-MB, and content of MDA (a typical marker of lipid peroxidation) in rats induced by ISO. CORT also prevented pathological myocardial injury in rats induced by ISO. Moreover, CORT attenuated the increased protein levels of GRP78, CHOP, and caspase-12, and reduced the increase of LC3-II, LC3-II/I, Beclin-1, and p62 in rats induced by ISO.ConclusionsThese data demonstrate that CORT can attenuate ISO-induced acute myocardial injury in rats likely by reducing lipid peroxidation, and inhibiting endoplasmic reticulum stress and autophagy. This supports CORT as a potentially being a new target for preventing and treating myocardial injury and its related disease.

Effect of Lysimachia ramosa Wall. Ex Duby and Its n-Butanol Fraction on Glycogen Content and Some Energy Related Enzymes in the Cestode, Raillietina echinobothrida

The current investigation aims to evaluate the effect of leaf extract of the anthelmintic medicinal plant Lysimachia ramosa and its n-butanol fraction on glycogen concentration and carbohydrate related metabolic enzymes viz. hexokinase (HK), phosphofructokinase, pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), malic enzyme (ME) and fumarate reductase (FR) in the cestode Raillietina echinobothrida. Following exposure to crude leaf extract and n-butanol fraction at a dose of 6 mg/ml of phosphate buffered saline, glycogen concentration decreased by 26–51% post paralysis whereas in terms of carbohydrate related metabolic enzymes, highest inhibition was seen in case of MDH (66–75%) followed by FR (64–66%), PEPCK (54–64%), LDH (48–60%), ME (33–38%) and PK (27–36%). The decrease in the enzyme activities of HK, LDH and MDH were also demonstrated histochemically. The result indicates that the intrusion of the phytoconstituents in the function of enzymes of energy generating pathway of R. echinobothrida, perhaps dispossesses the parasite from energy production leading to paralysis.

Does a cannibal feeding strategy impart differential metabolic performance in young burbot (Lota lota maculosa)?

The practice of mitigating cannibalism in aquaculture is an important focus for hatcheries seeking to maximize yield and has been maintained in hatcheries focusing on wild stock restoration. We hypothesize, however, that a cannibal feeding strategy may confer performance advantages over a non-cannibal feeding strategy and that perhaps cannibal size grading may not be optimal for hatcheries focusing on conservation goals. This study examined metabolic performance differences between cannibal and non-cannibal burbot, Lota lota maculosa, at the Kootenai Tribe of Idaho Twin Rivers Hatchery in Moyie Springs, ID, USA. After habitat alteration led to functional extinction of burbot in the region, the Twin Rivers Hatchery has played a leading role in the reestablishment of burbot in the Kootenai River, ID, and British Columbia. We examined morphometric data (weight, length and condition factor), whole animal resting metabolic rate and the enzyme activity of lactate dehydrogenase, citrate synthase and 3-hydroxyacyl-CoA dehydrogenase to describe the baseline metabolic performance of cannibal and non-cannibal burbot. Taken together, our results demonstrated significant differences in the metabolic strategies of cannibal vs. non-cannibal burbot, where cannibals relied more heavily on carbohydrate metabolism and non-cannibals relied more heavily on glycolytic and lipid metabolism. This study demonstrates the need to reevaluate the traditional practice of removing cannibal fish in conservation hatcheries, as it may not be the ideal strategy of raising the most robust individuals for release. When natural habitat conditions cannot be restored due to permanent habitat alteration, prioritizing release of higher performing individuals could help achieve conservation goals.

Study of the relationship between the age of the rams and the quality of their ejaculates obtained outside the breeding season

The present study aims to research the effect of age on the quality of ejaculates of rams from Synthetic population of Bulgarian dairy breed. Rams divided into two age groups were used for the experiment: G1 (2.5 years) versus G2 (6 years). Ejaculates were obtained by the method of artificial vagina and diluted with medium 6A-G. The tested ejaculates were obtained outside the breeding season. A Sperm Class Analyzer (SCA Microptic, Barcelona, Spain) was used to study sperm motility. The biochemical activity of the enzymes lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) was determined using an automatic Mindrai BA88 spectrophotometer. The activity of the enzymes was studied in sperm plasma, in aqueous and tritonic extract. Significant differences between the groups were found in total sperm motility (P?0.05) and LDH enzyme activity in sperm plasma (P?0.01). Regarding the efficiency of the LDH enzyme in aqueous and tritone extract, no significant differences between the groups was found. No significant differences were found between groups for GGT activity, while age had significant impact on LDH enzyme in sperm plasma.

Anticancer, Antiproliferative and Lactate Dehydrogenase Enzyme Activities of Different Parts of Acorn (Quercus coccifera L.) against A549, MCF-7 and HeLa Human Cancer Cells

Bu çalışmada, Meşe palamudunun (Q. coccifera) dış-kabuk, külah ve iç kısmından elde edilen özütlerin; A549, MCF-7, HeLa kanser hücreleri ve kanserli olmayan HUVEC hücrelerine karşı potansiyel antikanser, antiproliferatif ve nekrotik kapasiteleri araştırılmıştır. Analizler sonucunda, meşe palamudunun farklı kısımlarından elde edilen ekstrelerin en düşük konsantrasyonda dahi antikanser ve antiproliferatif etkiye sahip oldukları ortaya konulmuştur. Palamut meyvesinin külah kısmından elde edilen etanol özütleri, 5.04±0.02 ile 18.04±0.16 µg/mL arasında değişen IC50 değerleriyle, diğer kısımlarından elde edilen özütlerden daha yüksek antikanser aktivite göstermiştir. En yüksek oranda sitotoksik aktivite MCF-7 hücrelerine karşı gözlenirken; en düşük sitotoksik aktivite ise A549 hücrelerinde gözlenmiştir. Antikanser ve antiproliferatif aktivite sonuçlarının aksine, en yüksek LDH aktivitesi HeLa hücrelerinde belirlenmiştir. MTT ve tripan mavisi analiz sonuçlarıyla uyumlu olarak, en yüksek LDH salınım kapasitesi külah kısmından elde etanol özütlerinde saptanmıştır. Bu çalışma; meşe palamudunun, kanser hücrelerinde hücre büyümesini konsantrasyon ve zamana bağlı olarak engelleme potansiyeline sahip olduğunu ortaya koyan, özgün bir çalışmadır.

Effect of hesperetin on systemic inflammation and hepatic injury after blunt chest trauma in rats

ABSTRACTWe investigated the protective effect of hesperetin on hepatic damage after blunt chest trauma in rats using histological and biochemical methods. We used 18 adult male rats in three groups of six: control, chest trauma and chest trauma + hesperetin. Chest trauma was caused by dropping a metal cylinder onto the right hemithorax. Hesperetin, 100 mg/kg, was administered orally for 7 days. At the end of the seventh day, liver tissue samples were obtained. Serum tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), alanine aminotransferase (AST), aspartate transferase (ALT) and lactate dehydrogenase (LDH) enzyme activities were measured in blood samples taken from the heart. The general structure of liver tissue was investigated using hematoxylin and eosin staining. Nuclear factor kappa beta (Nf-κβ) expression in liver tissue was determined by the indirect immunohistochemical method. Apoptosis was determined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method. Decreased TNF-α, AST and ALT enzyme activity, fewer histopathological changes and lower Nf-kB expression were observed in the hesperetin treated group compared to the chest trauma group. We also found reduced hepatic apoptosis in the chest trauma + hesperetin group compared to the chest trauma group. Hesperetine inhibits liver damage by reducing proinflammatory cytokines and by suppressing Nf-κβ activity in a blunt chest trauma model in rats.

Гематологические параматеры молоди карпа на фоне введения в рацион экстракта коры дуба (Quercus Cortex)

The article presents the results of studies of morphological and biochemical blood parameters of carp juveniles when adding the extract Quercus cortex (1, 2, 3 mg/kg of feed), probiotic preparation of Soya-bifidum (0.7 ml/kg of feed), antibiotics Ciprofloxacin hydrochloride in the composition of the drug Antibak 250 (100 mg/kg of feed). Analysis of hematological parameters of carp juveniles showed that adding Quercus cortex extract to the diet at a dosage of 2 mg/kg of feed contributed to increasing SGE by 66%, the average concentration of hemoglobin in the red blood cell by 20% compared to the control group. An increase in the level of leukocytes compared to the control was recorded only in the IV experimental group and made 13% (P≤0.05). An increase in glucose in the groups receiving Quercus cortex extract has been stated: in the I experimental group- by 4.2 times (P≤0.001), in II – by 3.4 (P≤0.001) and in III – by 3.3 (P≤0.001) compared to the control. Increasing glucose concentration indicates active metabolic processes and better stress resistance in the fish body. Iron content in carp blood was higher only in the first experimental group (by 3 times) compared to the control. Increasing activity of alanine aminotransferase was observed in all experimental groups, the highest rate of this enzyme being recorded in the IV experimental group (feed with probiotic additive Soya-bifidum) with a significant excess of control by 48%. Lactate dehydrogenase enzyme activity exceeded the control in all experimental groups, maximum increase (by 67%) was observed in the IV experimental group. Alkaline phosphatase was below the control in all experimental groups, minimum values being registered in the IV experimental group- by 37% less than the control. Indicators of p-amylase in the IV group exceeded the control by 2.6 times, in the V group – by 1.5 times. In the II group p-amylase was lower than the control by 44%, and in the III – by 1.9 times. According to the obtained hematological data, it has been established that including Quercus cortex extract into the diet of carp juveniles is promising due 
 to the positive effect on the fish physiological status, immune status, metabolic processes, hematopoiesis and digestibility of feed.

Links between muscle phenotype and life history: differentiation of myosin heavy chain composition and muscle biochemistry in precocial and altricial pinniped pups.

In marine mammals, muscular development has been identified as a rate-limiting factor in achieving adult dive capacities. This study investigates the rate that myosin heavy chain (MHC) composition matures in a postural and locomotor skeletal muscle for four pinniped species with different lactation lengths: hooded seals, Cystophora cristata; harp seals, Pagophilus groenlandicus; northern fur seals, Callorhinus ursinus, and Steller sea lions, Eumetopias jubatus. The ontogeny of MHC isoform expression was compared with developmental rates of myoglobin concentrations, and aerobic (citrate synthase, β-hydroxyacyl-CoA dehydrogenase) and anaerobic (lactate dehydrogenase) enzyme activities. Within taxonomic families, species with shorter lactation periods had more mature muscles biochemically at birth, and fiber types differentiated earlier during ontogeny (Phocidae: hooded > harp seals, Otariidae: northern fur seals > Steller sea lions). Northern fur seal neonates had the most phenotypically-mature muscles in this study, with no immature MHC isoforms. The relationship between muscle biochemistry and MHC composition became more pronounced with age, and developed to reflect swimming mode and activity levels. In adults, phocids had more slow-twitch oxidative protein in their primary locomotor muscle, the Longissimus dorsi (LD), than otariids which likely reflects oxygen-sparing strategies for the phocids' longer dives. Conversely, northern fur seal muscles had higher proportions of fast-twitch MHCs in the Pectoralis and LD, likely indicative of this species' smaller size and higher mass-specific metabolic rates. Thus, muscle phenotype is linked with species life history, and a mismatch between muscle biochemistry and MHC composition at weaning has important implications for the first year of independent foraging in pinniped pups.

Withaferin A suppresses skin tumor promotion by inhibiting proteasome-dependent isocitrate dehydrogenase 1 degradation.

BackgroundThe metabolic enzyme isocitrate dehydrogenase 1 (IDH1) belonging to β-decarboxylase dehydrogenase family has been identified as a tumor suppressor. Withaferin A (WA), a bioactive compound derived from Withania somnifera, has the anti-tumor activity. Based on the data set that WA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IDH1 inactivation and mitochondrial dysfunction, we focused on how WA suppressed the skin carcinogenesis mediated by IDH1.MethodsThe mRNA levels of IDH1 were measured after treated with TPA and/or WA. The expression of IDH1, lactate dehydrogenase (LDH) involved in glycolysis, hypoxia inducible factor-1α (HIF-1α) and its target gene glucose transporter-1 (Glut1) were detected. The activities of proteasome and the mitochondrial complex I related to mitochondrial functions were determined. The enzymatic activities of LDH, proline hydroxylase (PHD) and vascular endothelial growth factor (VEGF) were analyzed.ResultsThe qPCR data have shown the mRNA levels of IDH1 were no difference with TPA and/or WA treatment. Next, data demonstrated that WA could stabilize IDH1 by inhibiting the ubiquitin-proteasome pathway (UPP). Followed by illuminating the mechanism of IDH1 inhibiting tumorigenesis, the results mirrored that upregulated IDH1 suppressed LDH activity whereas increased mitochondrial complex I activity. Furthermore, via its product α-KG, upregulated IDH1 activated PHD, and inhibited HIF-1α and its downstream signaling pathway.ConclusionsOur results indicate that WA inhibits tumor promotion partially via stabilizing IDH1, leading to inactivating the HIF-1α signaling.

Устойчивость к негативным воздействиям и соотношение активности ферментов энергетического обмена в тканях черноморских моллюсков Mytilus galloprovincialis Lamarck, 1819 и Anadara kagoshimensis (Tokunaga, 1906)

Определение соотношения активности ферментов энергетического обмена малатдегидрогеназы (МДГ, 1.1.1.37) и лактатдегидрогеназы (ЛДГ, 1.1.1.27) позволяет получить интегральную оценку физиологического состояния объекта исследования в ответ на воздействия разной природы. Цель работы ― сравнить изменение величины отношения МДГ/ЛДГ в тканях двустворчатых моллюсков: аборигенной Mytilus galloprovincialis Lamarck, 1819 и успешного вселенца Anadara kagoshimensis (Tokunaga, 1906), в лабораторных условиях под влиянием гипоксии, аноксии, токсиканта полихлорбифенила, сероводородного заражения и длительного содержания в аквариуме без кормления. Половозрелые моллюски собраны в районе г. Севастополя. Длина раковины мидии составляла 45–62 мм, анадары ― 27–49 мм. Активность ферментов измеряли спектрофотометрически (при 340 нм и 25 °C) по скорости окисления НАДН в цитоплазме тканей (мышцы, гепатопанкреас, жабры). Как правило, под воздействием негативных факторов активность ЛДГ снижалась значительно (на 36–80 %), активность МДГ оставалась стабильной, а коэффициент МДГ/ЛДГ в тканях обоих видов моллюсков увеличивался в 1,5–4 раза. Отмечено, что у гемоглобинсодержащей анадары коэффициент МДГ/ЛДГ был на порядок ниже, чем у мидии, как в контроле, так и в опыте. Сопоставление с литературными данными показало, что величина отношения МДГ/ЛДГ устойчивой к гипоксии анадары такая же низкая, как у оксифильных гидробионтов, в частности гребешка Mizuhopecten yessoensis (Jay, 1857) и ракообразных Crangon crangon (Linnaeus, 1758) и Carcinus aestuarii Nardo, 1847. По-видимому, низкое значение коэффициента МДГ/ЛДГ отражает способность анадары сохранять высокий уровень окислительных процессов в тканях благодаря значительному пулу соединений (эритроцитарный гемоглобин, каротиноиды, глутатион), поддерживающих аэробный процесс и осуществляющих антиоксидантную защиту. Величина коэффициента МДГ/ЛДГ может быть использована для оценки степени оксигенации тканей моллюсков в норме и в условиях гипоксии различного происхождения при мониторинговых исследованиях.

Studying the Effect of Some Thiadiazole Derivatives on the Activity of LDH Enzyme

This work involved studying the effect of some thiadiazole derivatives on the activity of lactate dehydrogenase enzyme (LDH) in the serum of normal control and thalassemia patients. The results showed that all the compounds used had inhibitory effect on LDH enzyme activity, the inhibition percent was between (7.87%-55.22%) depending on compound structure and its concentration. Studying kinetic properties showed that these compounds behave as non-competitive inhibitors.

Abstract 3082: Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect. Citation Format: Btissame El Hassouni, Rocco Sciarrillo, Valentina Edith Gómez, Mina Maftouh, Giulia Mantini, Christian M. Vonk, Carlotta Granchi, Niccola Funel, Filippo Minutolo, Godefridus J. Peters, Elisa Giovannetti. Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3082.

Immunomodulatory and antioxidant effects of pomegranate peel polysaccharides on immunosuppressed mice

This work aims to analyze the immunomodulatory effect of pomegranate peel polysaccharides (PPP) on the immunosuppressed mice induced by cyclophosphamide (CTX). All the mice were divided into 6 groups randomly and the immunoprophylaxis mice were administrated with PPP [100, 200, 400 mg/(kg·d)] by gavage for consecutive 28 days. The results showed that PPP can slow down the decline of body weight and increase the immune organ index of the immunosuppressed mice. Compared to the model mice, the enzymatic activity of LDH (lactate dehydrogenase) and ACP (acid phosphatase) of the mice spleen administrated with PPP by gavage was enhanced significantly. PPP stimulated proliferation and secretion of splenic lymphocytes and markedly increased the immunoglobulin (Ig-A, Ig-G and Ig-M) expression and the release of cytokines (TNF-α, IL-2 and INF-γ) in cyclophosphamide-induced immunosuppressed mice. Hepatic antioxidant enzymatic activities of T-AOC (total antioxidant capacity), T-SOD (total superoxide dismutase), GSH-PX (glutathione peroxidase) and CAT (catalase) were markedly increased when the mice were administrated with high dosage of PPP. So it can be concluded that PPP could be used as an efficacious adjacent immunopotentiating therapy or an alternative means in lessening chemotherapy-induced immunosuppression, and also can be utilized as immunostimulants for food and pharmaceutical industries.

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms

SummaryLate embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11‐amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α‐helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2O2), suggesting a protective role in a chaperone‐like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress‐response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.

The surface markers and survival rate of platelets during storage at 4°C: The influence of sodium octanoate

Background: Storage of platelet concentrates (PCs) at room temperature (20-24°C) limits its storage time to 5 days due to the destructive effects of platelet storage lesion (PSL) and bacterial contamination. Although prolonged storage of platelets (PLTs) at 4°C reduces the likelihood of bacterial contamination and PSL levels, it is accompanied by an increase in the clearance rate and changes in the surface markers of PLTs. The goal of this study was to evaluate the effects of sodium octanoate (SO) as a stabilizer on PLTs during storage at 4°C. Materials and Methods: In this experimental study, PCs were divided into three portions and stored for 5 days at 3 different conditions, including 20-24°C, 4°C temperature, and 4°C in presence of SO. PLTs enumeration was performed using an automated hematology analyzer. To measure the metabolic activity and survival rate of PLTs, the water-soluble tetrazolium salt (WST-1) assay was performed. The activity of lactate dehydrogenase enzyme (LDH) was measured by a biochemical analyzer. Additionally, the levels of PLT glycoprotein Ibα (GPIbα) and CD62P (P-selectin) were measured on PLTs by flow cytometry technique. Results: PLTs count was higher in SO-treated (4°C) PLTs than two other studied samples. Additionally, the viability was higher in the SO-treated PLTs than that in other groups. LDH amount was lower in the SO-treated PLTs than that in other groups (P>0.05). GPIbα expression was significantly higher in SO-treated PLTs than that other groups (P<0.05). On the other hand, the expression of CD62P was lower at 4°C in PLTs in the presence of SO (P>0.05). Conclusions: SO could modulate the effects of cold temperatures on PLTs. Furthermore, we found that the survival of platelets was better maintained in the presence of SO at 4°C.

Scopoletin and Morin Inhibit Lactate Dehydrogenase Enzyme Activity, Which Is Critical for Cancer Metabolism

Background: Lactate dehydrogenase (LDH) is a tetrameric enzyme that catalyzes the interconversion of pyruvate to L-lactate. The importance of this enzyme is because LDH isoenzymes are involved in cancer, heart, and liver diseases. Inhibition of this enzyme can help prevent and treat different diseases. Morin is a flavonoid found in the Moraceae family and scopoletin is a coumarin found in Scopolia genus. Objectives: The aim of this study was to determine the effect of morin and scopoletin as two natural products on the activity and structure of lactate dehydrogenase enzyme. Methods: Morin and scopoletin were examined for inhibition of the activity of LDH in 100 mM sodium phosphate buffer pH 7.5, at room temperature using UV-V spectrophotometry. Fluorescence spectroscopy was used to characterize protein structural changes in the presence of morin and scopoletin. Results: Km and Vmax of LDH for pyruvate were 11.69 mM and 1.258 mM/min, respectively. The kinetic results showed that morin and scopoletin are LDH inhibitors. The Ki values of morin and scopoletin were determined as 1.78 µM and 0.8 µM, respectively, using a secondary plot. Fluorescence intensity quenching and red shift of the maximum wavelength of emission in a concentrationdependent manner showed that morin and scopoletin bind to LDH and affect its structure. Conclusions: The results suggest that morin and scopoletin bind to LDH, influence its conformation and inhibit its activity. Scopoletin showed more effective inhibition of LDH activity and it can be a promising candidate in the field of tumor metabolism inhibitors.

LABORATORY CRITERIA OF PERINATAL DAMAGE OF CENTRAL NERVOUS SYSTEM AT PREMATURE NEWBORNS

Fetal and neonatal hypoxia takes a special place among the damaging factors of central nervous system (CNS). All forms of oxygen deficiency are accompanied by the development of bioenergetic hypoxia, which leads to tension of metabolic processes of the organism. Metabolic effect of hypoxia includes stark reduce of mitochondrial activity due to a significant inhibition enzymes of the Krebs cycle: succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH). In newborn babies is not always possible to objectively assess the condition of the CNS defeat, because very often the severity of lesions does not correspond to clinical symptoms, especially in premature newborns. So far determination the severity of hypoxic-ischemic CNS lesions is still very actual in modern medicine. More objective method of such an assessment is determine the activity of neurospecific enolase (NSE). The aim of the paper is to increase the efficiency of diagnosis of hypoxic CNS lesions in premature infants by determining the activity of NSE and study energy supply during the neonatal period. The concentration of NSE, SDH and LDH were determined in 15 conventionally healthy preterm infants (CHPI), which made the comparison group, and 64 premature babies with hypoxic-ischemic CNS lesions, which were divided into three groups: I group – 26 premature children with mild CNS lesions; II group – 20 premature children with severe hypoxic lesions and low birth weight; III group – 18 premature newborns with severe damage of central nervous system and extremely low birth weight. NSE activity was determined by enzyme immunoassay using reagents of the company «Fujirebio» (Sweden) on an automatic analyzer «Multiscan Plus» company «Labsystems» (Finland). Material for investigation was peripheral venous blood of newborns, which collected by vein punction at morning on an empty stomach. Metabolic effect of hypoxia in premature infants manifested by severe inhibition of mitochondrial respiratory activity, which appears in the reduction of aerobic enzyme activity of SDH and activation serum LDH. During the neonatal period in infants with perinatal hypoxic- ischemic lesions of the CNS levels of the of NSE, SDH and LDG aren’t normalized, that indicated on energy deficiency and requires the development of effective methods of correcting this condition. Perinatal hypoxia in premature neonates causes significant alteration of neuronal membranes and increase concentration in blood such neurospecific protein as NSE, whose concentration correlates with the degree of severity of CNS injury.

Voluntary wheel running prevents the acidosis-induced decrease in skeletal muscle mitochondrial reactive oxygen species emission.

Decreases in pH (acidosis) in vitro can alter skeletal muscle mitochondrial function [respiration and reactive oxygen species (ROS) emission]. However, because skeletal muscles readily adapt to exercise, the effects of acidosis may be different on sedentary vs. trained muscle. The aim of this work was to compare the effects of pH on skeletal muscle mitochondrial function between sedentary vs. exercise-trained male Sprague-Dawley rats ( n = 10 in each cohort). Rates of mitochondrial respiration and ROS emission were determined from the soleus muscle of both cohorts over a physiologic range of pH values (pH 6.2-7.1). Exercise-trained rats had 14% higher mean muscle buffering capacities; 46 and 40% greater enzyme activity of citrate synthase and lactate dehydrogenase, respectively; and greater activity of respiratory complexes I-IV. ADP-stimulated respiration with complex I and II substrates was ∼25% greater in exercise-trained rats but was unaffected by pH in either cohort. In both cohorts, lowering pH decreased respiration only in complex I- and complex II-supported nonphosphorylating (leak) state. However, as pH decreased, ROS emissions in complex I- and complex II-supported leak state decreased only in sedentary rats; in exercise-trained rats, ROS emissions in this state remained constant. We hypothesize that this effect may result from modulation at complex III, which declined 47% per unit pH in sedentary rats, in comparison to 23% in exercise-trained rats. Taken together, these data suggest that pH regulates mitochondrial respiratory complexes and that exercise training can decrease the effects of pH on skeletal muscle mitochondrial function.-Hedges, C. P., Bishop, D. J., Hickey, A. J. R. Voluntary wheel running prevents the acidosis-induced decrease in skeletal muscle mitochondrial reactive oxygen species emission.

Designing the Optimal Formulation for Biopharmaceuticals: A New Approach Combining Molecular Dynamics and Experiments

Biopharmaceuticals are often stored in a lyophilized form. However, stresses due to both the freezing and the drying steps of the lyophilization process can be harmful to protein stability, and appropriate excipients must be added to minimize detrimental effects. In this work, molecular dynamics was used to provide insight into the mechanisms of protein stabilization by different osmolytes, using lactate dehydrogenase as model protein. Our simulations indicate that good cryoprotectants are not always equally good as lyoprotectants, suggesting that synergistic effects may arise when different excipients are combined. This observation is in accordance with the experimental results. In fact, the enzymatic activity of lactate dehydrogenase after freeze-drying was investigated for various formulations, and the trend predicted by molecular dynamics was confirmed. More specifically, we found that the most effective stabilization of the protein structure is achieved when a good cryoprotectant is coupled with an efficient lyoprotectant. Ultimately, we propose a new approach to the design of formulations for protein-based therapeutics to be lyophilized, which combines simulations and experiments. In this new concept, the computational investigation allows a more knowledge-driven and targeted experimental campaign for the selection of the optimal excipients, making the whole process extremely time- and cost-effective.