Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells.

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

Circ-PRMT5 enhances the proliferation, migration and glycolysis of hepatoma cells by targeting miR-188-5p/HK2 axis.

INTRODUCTION AND OBJECTIVES: Circular RNA (circRNA) has been demonstrated as a critical regulator in human cancer, including hepatocellular carcinoma (HCC). Nevertheless, the role of circ-PRMT5 in HCC remains largely unknown. PATIENTS OR MATERIALS AND METHODS: The real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression levels of circ-PRMT5, miR-188-5p and anti-Hexokinase II (HK2) in HCC tissues and cells. The cell proliferation, migration and glycolysis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell migration assay, and indicated kits, respectively. The interaction relationship between miR-188-5p and circ-PRMT5 or HK2 was analyzed by the bioinformatics database, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) assay. The western blot assay was used to analyze the expression level of HK2. The functional role of circ-PRMT5 in vivo was assessed by a xenograft experiment. RESULTS: Circ-PRMT5 was elevated in HCC tissues and cells than matched control groups. Furthermore, loss-of-functional experiments revealed that the silencing of circ-PRMT5 could repress proliferation, migration, glycolysis in vitro and tumor growth in vivo. Moreover, we also confirmed that overexpression of circ-PRMT5 abolished the effects on HCC cells induced by upregulating miR-188-5p. In addition, overexpression of miR-188-5p could repress the development of HCC. More importantly, HK2 was a target gene of miR-188-5p, and miR-188-5p regulated proliferation, migration, glycolysis of HCC cells by specifically binding to HK2. Mechanistically, circ-PRMT5 could act as a sponge of miR-188-5p to regulate the expression of HK2. CONCLUSION: In summary, circ-PRMT5 might play a key role in proliferation, migration, glycolysis of HCC cells via miR-188-5p/HK2 axis, which indicated that circ-PRMT5 might be a potential therapeutic target for HCC treatment.

Effects of copper toxicity at different pH and temperatures on the in vitro enzyme activity in blood and liver of fish, Prochilodus lineatus.

Blood and liver from curimbata (Prochilodus lineatus) acclimated at pH 4.5, 7.0 and 8.0 and at 20 and 30 °C were exposed in vitro to different concentrations of copper (Cu): 98 ± 0.8 µg Cu L-1 at pH 4.5 and 16 ± 0.2 µg Cu L-1 at pH 8.0 at 20 °C; 88 ± 0.8 µg Cu L-1 at pH 4.5 and 14 ± 0.5 µg Cu L-1 at pH 8.0 at 30 °C and in 29 µg Cu L-1 at pH 7.0 at 20 and 30 °C for 2 h. The pH affected the levels of glucose and glycogen and in vitro exposure to Cu increased glucose levels and decreased glycogen at 20 and 30 °C. Exposures to acid water and Cu in vitro also resulted in an increase in enzyme activity in the blood, hexokinase (HK), pyruvate kinase (PK) and at pH 8.0 decreased the HK activity and increased PK and lactate dehydrogenase (LDH) activities at 20 °C. Cu caused an increase in the activities of HK (at pH 4.5) and PK in both pH, 4.5 and 8.0 at 30 °C. At 20 °C, HK (pH 8.0) and glycose-6-phosphate dehydrogenase (G6PDH) (pH 4.5) activities increased and PK (pHs 4.5 and 8.0) and LDH decreased (pH 4.5) in the liver. In vitro, exposure to Cu increased HK and G6PDH at pH 8.0 and PK activity increased in both pH values and LDH increased in pH 7.0. Cu exposure in vitro at 30 °C, phosphofructokinase (PFK) and PK activities decreased and LDH activity increased in all pH values when compared to fish from the water from its respective pH values. Interactions occurred in blood and liver between the temperature, pH and exposure to copper in vitro. The in vitro tests may constitute an interesting biological model for experimental and applied toxicology, especially in the case of environmental pollution.

Essential oil of Cymbopogon citratus (lemongrass) and geraniol, but not citral, promote gastric healing activity in mice.

Cymbopogon citratus, popularly known as lemongrass, is used for the treatment of gastric, nervous and hypertensive disorders, in addition to its use in the food and pharmaceutical industries. This study evaluated the gastroprotective and gastric healing effect of essential oil of C. citratus (EOCC), citral and geraniol at doses of 1-100 mg/kg (p.o) on acute ethanol-induced ulcer and chronic acetic acid-induced ulcer. Histological and histochemical evaluation was also performed, as well as the in vitro evaluation of the effects of these phytochemicals on H+/K+-ATPase activity. In the ethanol-induced gastric ulcer, the minimum effective oral dose of EOCC, citral and geraniol were 10, 100 and 3 mg/kg, reducing the ulcer area by 51.67%, 96.57% and 55.74%, respectively, compared to vehicle group (25.82 ±â€¯3.59 mm2). Moreover, EOCC (10 mg/kg, p.o) and geraniol (3 mg/kg), but not citral (100 mg/kg), accelerated the gastric healing process by 34.52 and 80.57%, compared to acetic-acid ulcerated group treated with vehicle (36.04 ±â€¯1.03 mm2). These healing effects were confirmed histologically by the contraction of the ulcer base and by the enhancement on mucin staining in slices of ulcer site from mice treated with EOCC or geraniol. Interestingly, EOCC and citral at 100 µg/ml inhibited the H+/ K+-ATPase activity by 28.26% and 44.36%, whereas geraniol did not change this parameter. Together, these findings confirm the gastroprotective and healing gastric ulcer effects of essential oil from aerial parts of C. citratus and added the information that geraniol, but not citral, promotes healing effects on installed ulcers.

Disección Submucosal Endoscópica con Hibrid-Knife: Experiencia Preliminar en Latinoamérica / Endoscopic submucosal dissection Hybrid Knife: Preliminary experience in Latin American

Introducción: La disección submucosal endoscópica con Hybrid Knife (DSEH) es una técnica prometedora para la resección de tumores en etapa temprana. Hay poca data en Latinoamérica. Pacientes y métodos: Estudio prospectivo-descriptivo (marzo 2011 - marzo 2012). Se incluyeron 25 pacientes (16 hombres, 9 mujeres), edades comprendidas entre 52-72 años (X=62,52 años). Se realizaron 25 procedimientos DSEH. Las indicaciones fueron: tumores subepiteliales (7), neoplasia de colon y recto (16), neoplasia precoz gástrica (2). Resultados: DSEH fue técnicamente posible en todas (25) las lesiones (100%). La resección en bloque y márgenes libres de lesión se obtuvieron en todos los casos. El tamaño de la mucosa disecada fue entre 2-7 cms (X=3,8 cms). El tiempo endoscópico fue entre 45-120 minutos(X=84,4 minutos). Perforación ocurrió en 2 casos, siendo resuelta con tratamiento endoscópico (clips). Mortalidad no fue reportada. Conclusiones: Los resultados preliminares sugieren que la DSE con Hibrid Knife (DSEH), parece ser una buena opción para el tratamiento endoscópico de tumores en etapa temprana gástricos, recto colónico y tumores carcinoides. Estudios controlados, aleatorizados de la DSE con Hibrid Knife, en comparación con otros dispositivos son necesarios.

Introduction: Endoscopic submucosal dissection Hybrid Knife (ESD-HK) is a promising technique for resection of early stage tumors. Few data in Latin America. Patients and methods: Prospective, descriptive study (March 2011-2012). 25 patients (16 men, 9 women), mean age 62.52 years (52-72 years).25 procedures were performed. Indications: sub-epithelial tumors (7), colorectal neoplasia (16) early gastric neoplasia (2) Results: ESD-HK was technically possible in all (25) lesions (100%). En bloc resection and free margins were obtained in all cases. The diameter of dissected mucosa was between 2-7 cms(X=3.8 cms) The time was between 45-120 minutes(X= 84.4 minutes). Perforation occurred in 2 cases being resolved with endoscopic treatment (clips). Mortality was not reported. Conclusions: Our preliminary results suggest that the DSE with Hibrid Knife (DSEH) seems to be a good option for endoscopic treatment of early stage gastric tumors, colon and rectal carcinoid tumors. Studies controlled, randomized DSE with Hibrid Knife, compared with other devices are needed.

Expression of Genes Involved in Porphyrin Biosynthesis Pathway in the Human Renal Cell Carcinoma.

Renal cell carcinoma (RCC) remains one of the greatest challenges of urological oncology and is the third leading cause of death in genitourinary cancers. Surgery may be curative when patients present with localized disease. Our previous results demonstrated the autofluorescence of blood PpIX in primary RCC mouse model and an increase in fluorescence intensity as a function of growth of the subcutaneous tumor mass. In another work, a nice correlation between the growth of the tumor mass and tissue fluorescence intensity was found. The aim of this study was to evaluate the expression profile of porphyrin biosynthesis pathway-related genes of human kidney cells. We used two kidney cell lines, one normal (HK2) and another malignant (Caki-1). Endogenous and 5-aminolevolinic acid (ALA) induced protoporphyrin IX (PpIX) HK2 and Caki-1 cells were analyzed by fluorescence spectroscopy. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure mRNA of those genes. Emission spectra were obtained by exciting the samples at 405 nm. For ALA untreated cells the maximum fluorescence intensity was detected at 635 nm. The mean peak area of emission spectra in both cells types increased linearly in function of cell number. Besides, basal levels of PpIX autofluorescence of each cell concentration of HK2 samples were significantly lower than those of Caki-1 samples. For ALA-treated cells the mean PpIX spectra shows PpIX emission peak at 635 nm with a shoulder at 700 nm. Analysis of PpIX fluorescence intensity ratio between tumor cells and HK2 cells showed that fluorescence intensity was, on average, 26 times greater in tumor cells than in healthy cells. qRT-PCR revealed that in Caki-1 ALA-treated cells, PEPT gene was significantly up-regulated and FECH and HO-1 genes were significantly down regulated in comparison with HK2 ALA-treated cells. In conclusion, our results demonstrate the preferential accumulation of ALA-induced PpIX in human RCC and also indicate that PEPT1, FECH and HO-1 genes are major contributors to this accumulation.

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes.

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and -0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.

Family-based association study between SLC2A1, HK1, and LEPR polymorphisms with myelomeningocele in Chile.

Obese/diabetic mothers present a higher risk to develop offspring with myelomeningocele (MM), evidence supporting the role of energy homeostasis-related genes in neural tube defects. Using polymerase chain reaction-restriction fragment length polymorphism, we have genotyped SLC2A1, HK1, and LEPR single-nucleotide polymorphisms in 105 Chilean patients with MM and their parents in order to evaluate allele-phenotype associations by means of allele/haplotype transmission test (TDT) and parent-of-origin effects. We detected an undertransmission for the SLC2A1 haplotype T-A (rs710218-rs2229682; P = .040), which was not significant when only lower MM (90% of the cases) was analyzed. In addition, the leptin receptor rs1137100 G allele showed a significant increase in the risk of MM for maternal-derived alleles in the whole sample (2.43-fold; P = .038) and in lower MM (3.20-fold; P = .014). Our results support the role of genes involved in energy homeostasis in the risk of developing MM, thus sustaining the hypothesis of diverse pathways and genetic mechanisms acting in the expression of such birth defect.

Polyphenols of Mangifera indica modulate arsenite-induced cytotoxicity in a human proximal tubule cell line

Inorganic arsenic is an ubiquitous environmental contaminant able to cause severe pathologies in humans, including kidney disorders. The possible protective effects of Mangifera indica L., Anacardiaceae, stem bark extract (MSBE) and some mango phenols on the cytotoxicity of arsenite (AsIII) in the proximal tubule cell line HK-2 was investigated. In cells cultured for 24 h in presence of AsIII, a dose-dependent loss of cell viability occurred that was significantly alleviated by MSBE, followed by gallic acid, catechin and mangiferin. Mangiferin complexed with Fe+++ proved more efficacious than mangiferin alone. MSBE and pure phenols increased significantly the cell surviving fraction in clonogenic assays. In cells pretreated with MSBE or phenols for 72 h the protection afforded by MSBE resulted decreased in comparison with the shorter experiments. Cells pretreated with a subcytotoxic amount of AsIII or cultured in continuous presence of low concentration of mangiferin proved to be more resistant to AsIII, while cells cultured in presence of albumin resulted more sensitive. Because all the above conditions share changes in expression/activity of P-glycoprotein (P-gp), a transporter potentially involved in arsenic resistance, the capability of M. indica phenols in modulating AsIII-induced cytotoxicity would be at least in part dependent on their interactions with P-gp.

Salicylic acid degradation from aqueous solutions using Pseudomonas fluorescens HK44: parameters studies and application tools

The optimal conditions for salicylic acid biodegradation by Pseudomonas fluorescens HK44 were determined in this study with the intention to create a microbial sensor. Kinetic experiments permitted a definition of 60 and 30min the time needed to achieve the maximum degradation of salicylic acid presented in a medium with and without yeast extract, respectively. The degradation in medium without yeast extract and the quantification by spectrophotometry 230 nm were selected to be used in further tests. The use of preactivated cells or on the exponential growth phase showed better salicylic acid degradation percentages when compared to nonactivated cells or on the stationary growth state. Finally, the best cellular concentration used on the salicylic acid degradation was 0,1 g.L-1. Strain HK44 shows to be capable of degrade salicylic acid presented in simple aqueous systems, making this strain a promising tool for the application on a luminescent microbial sensor.

Com a intenção de criar um sensor microbiano, as condições ótimas para a biodegradação de ácido salicílico por Pseudomonas fluorescens HK44 foram determinadas neste estudo. Os experimentos cinéticos permitiram a definição dos tempos de 60 e 30 minutos como necessários para atingir a máxima degradação de ácido salicílico presente em meio com ou sem extrato de lêvedo, respectivamente. A degradação no meio sem extrato de lêvedo e a quantificação através de espectrofotometria 230 nm foram selecionadas para serem utilizadas em testes posteriores. O uso de células pré-ativadas ou na fase exponencial de crescimento apresentou melhores porcentagens de degradação de ácido salicílico quando comparadas a células não-ativadas ou no estado estacionário de crescimento. Além disso, a melhor concentração celular utilizada nessa degradação foi 0,1 g.L¹. A cepa HK44 parece ser capaz de degradar o ácido salicílico presente em sistemas aquosos simples, tornando este microrganismo uma ferramenta promissora para aplicação em um sensor microbiano luminescente.

The protective effects of Baypamun®HK in mice experimentally infected with Toxoplasma gondii / Efeitos do Baypamun®HK em camundongos infectados experimentalmente com Toxoplasma gondii

The present work aimed to verify the effects of Baypamun®HK on the survival of albinic mice, experimentally infected with Toxoplasma gondii RH strain, treated or not with sulfadiazine-pirimetamine, as well as on the formation and the number of brain cysts. Four groups of 20 mices were inoculated with 105 tachizoites, subcutaneously, and undergone to different treatments. Serum tests were carried out by indirect immunofluorescence and samples of brain, liver and lung were collected from animals who died for cytological and brain cysts examinations. After 60 days, all survivors were sacrificed. Isolated Baypamun®HK didn't present a protective action against the infection by T. gondii, but its association sulfadiazine-pirimetamine, a specific treatment caused a higher survival of animals and a more intense and longer antibody responses. (AU)

O presente trabalho teve como objetivo verificar os efeitos do Baypamun®HK sobre a sobrevida de camundongos albinos, experimentalmente infectados com Toxoplasma gondii amostra RH, submetidos ou não ao tratamento com sulfadiazina-pirimetamina, bem como sobre a formação e número de cistos cerebrais. Quatro grupos de 20 camundongos foram inoculados com 105 taquizoítos, via subcutânea, e submetidos a diferentes tratamentos. Os testes sorológicos foram realizados pela técnica de imunofluorescência indireta, e, dos animais que sucumbiram, amostras de cérebro, pulmão e fígado foram retiradas para exame citológico e de cérebros para a pesquisa de cistos. Ao final de 60 dias todos os sobreviventes foram sacrificados. Isoladamente o Baypamun®HK não apresentou uma ação protetora contra a infecção pelo T. gondii, mas sua associação com o tratamento específico pela sulfadiazina-pirimetamina promoveu maior sobrevida dos animais e uma resposta de anticorpos mais intensa e duradoura. (AU)