ERK 1/2 activation does not contribute to increased survival mediated by bone marrow cells after 90% partial hepatectomy in Wistar rats

We investigated the influence of bone marrow cells upon activation of ERK 1/2 in an animal model of 90% PH. Phosphorylated ERK 1/2 was evaluated by western blot. No differences were found between the groups. Thus, increased survival does not appear to be mediated by ERK 1/2 activation (AU)

New frontiers in the study of memory mechanisms

We review recent work on three major lines of memory research: a) the possible role of the protein kinase M-zeta (PKMzeta) in memory persistence; b) the processes of “synaptic tagging and capture” in memory formation; c) the modulation of extinction learning, widely used in the psychotherapy of fear memories under the name of “exposure therapy”. PKMzeta is a form of protein kinase C (PKC) that apparently remains stimulated for months after the consolidation of a given memory. Synaptic tagging is a mechanism whereby the weak activation of one synapse can tag it with a protein so other synapses in the same cell can reactivate it by producing other proteins that bind to the tag. Extinction, once mistakenly labeled as a form of forgetting, is by itself a form of learning; through it animals can learn to inhibit a response. We now know it can be modulated by neurotransmitters or by synaptic tagging, which should enable better control of its clinical use.

Fisiopatología de la retinopatía y nefropatía diabéticas / Pathophysiology of diabetic retinopathy and nephropathy

Despite the availability of multiple therapeutic approaches, diabetes mellitus with chronic hyperglycemia remains as the main cause of new cases of blindness and chronic renal failure in the western hemisphere. We herein review the molecular mechanisms by which chronic hyperglycemia causes retinopathy and nephropathy in type I and type 2 diabetic patients. Diabetic retinopathy develops silently along years or decades, producing symptoms only in its very ¡ate stages. Its slow development starts with the activation of aldose reducíase, shortly followed by the destruction of the retinal pericyte cells, and ends in sudden blindness when vitreous hemorrhage ensues. Nephropathy, on the other hand, centers its pathophysiology in the mesangial cell, that starts as a modified smooth-muscle cell, and turns itself into a myo-fibroblast, produces such amounts of cytoplasm and extracellular protein that strangulates the glomerular capillaries and causes renal failure. After a detailed review of the molecular mechanisms of the aforementioned complications, we conclude that, apart from directing our attention to the emerging medications that are being developed to block these molecular pathways, we should never abandon the struggle for improving the glycemic control of our diabetic patients.

Efeito da autofagia sobre a capacidade fagocítica e sobre a infecção de macrófagos de camundongos CBA/J por Leishmania amazonensis / Effect of autophagy on phagocytic capacity and the infection of macrophages from CBA /J mice by Leishmania amazonensis

A autofagia vem sendo alvo de estudos que demonstram sua participação em infecções por diversos patógenos intracelulares. A depender do patógeno, a autofagia pode facilitar a sobrevivência intracelular do patógeno ou pode funcionar como controle da infecção pela célula hospedeira. Pouco se sabe sobre a participação da autofagia na infecção por Leishmanía. Foi demonstrado que o vacúolo parasitóforo induzido por L. mexícana adquire nutrientes citosólicos por microautofagia. Além disso, recentemente foi demonstrado que a indução de autofagia promove aumento da carga parasitária de L. amazonensís em macrófagos infectados. Esses dados sugerem a participação do processo autofágico no estabelecimento da infecção por Leishmanía, como um mecanismo que favorece a sobrevivência intracelular do parasito. Assim, o objetivo desse estudo foi determinar a influência da autofagia na infecção, in vitro, de macrófagos de camundongos CBA/J por L. amazonensís. Macrófagos foram induzidos à autofagia por duas formas, fisiológica ou farmacológica, após ou antes da infecção por L. amazonensís ou exposição a partículas de levedo ou zimosan. O percentual de infecção e de fagocitose foi estimado. Os resultados mostram que a indução de autofagia, após a infecção, não altera o percentual de macrófagos infectados, mas promove o aumento na carga parasitária de macrófagos infectados por L. amazonensís. Além disso, a prévia indução de autofagia promove a inibição da capacidade fagocítica do macrófago murino. Estudos adicionais serão realizados no intuito de esclarecer os mecanismos pelos quais a indução de autofagia favorece a infecção por L. amazonensís e altera a capacidade fagocítica do macrófago murino.

Efeito da remoção de células somáticas pela microfiltração sobre a lipólise do leite / Effect of somatic cell removal by microfiltration on the lipolysis of milk

O objetivo do presente estudo foi avaliar o efeito da retirada mecânicadas células somáticas do leite cru sobre a taxa de lipólise durante o armazenamento refrigerado do leite pasteurizado. O delineamento experimental utilizado foi totalmente casualizado, com três repetições e arranjo fatorial de tratamentos 2 X 2 X 2, constituídos por dois nível de gordura do leite (desnatado e integral), dois níveis de contagem de células somáticas (CCS baixa - CCSB e CCS alta - CCSA), e pela aplicação ou não da microfiltração ao leite. Três lotes de leite cru CCSB (75.000 cél./mL) e CCSA (1.150.000 cél./mL) foram obtidos de vacas selecionadas e submetidos ao desnate centrífugo, à microfiltração em sistema a vácuo, sendo em seguida pasteurizados e armazenados por 21 dias sob refrigeração a 6ºC. Foram realizadas medidas repetidas no tempo, as quais corresponderam aos dias de coleta do leite pasteurizado durante o período de armazenamento (1, 7, 14 e 21). A microfiltração associada ao desnate foram eficazes na remoção das células somáticas do leite, com reduções de 92,5 e 99,5% para o leite CCSB e CCSA, respectivamente. A lipólise do leite aumentou em função do tempo de armazenamento e foi maior para o leite microfiltrado, contudo não sofreu influência da CCS. Independentemente da CCS, ocorreu aumento da lipólise do leite pasteurizado durante o período de armazenamento.

The objective of this study was to evaluate the effects of raw milk somatic cell removal by microfiltration on lipolysis of pasteurized milk during refrigerated storage. A completely randomized design was used, with 3 repetitions and a 2 X 2 X 2 factorial arrangement of treatments as follow: milk fat level (skimmed and whole milk), two different levels of somatic cell counts - low somatic cell count (LSCC) and high somatic cell count (HSCC) - and, use of microfiltration ornot. LSCC raw milk - 75,000 cells/ml - and HSCC raw milk - 1,150,000 cells/ml - were obtained from selected cows, skimmed and submitted to vacuum microfiltration. Milk from all treatments was pasteurized and kept refrigerated at 6ºC for 21 days. Repeated measures during storage time were taken from pasteurized milk at days 1, 7, 14 and 21. The application of milk microfiltration was efficient on somatic cell removal, with reduction of 92,5 and 99,5% for LSCC and HSCC,repectively. Lipolysis of milk was increased during storage period and was higher for milk submitted to icrofiltration, however no effect of SCC was observed. Lipolysis in pasteurized milk increased independently of SCC, during its refrigerated storage period.

Rapid regulatory effect of tri-iodothyronine (T3) on antioxidant enzyme activities in a fish Anabas testudineus (Bloch): short-term in vivo and in vitro study.

The short-term action of thyroid hormone tri-iodothyronine (T3) was studied in vivo and in vitro on antioxidant enzyme activities in a teleost Anabas testudineus (Bloch). T3 injection in vivo (200 ng) in normal fish decreased the lipid peroxidation products and increased superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities after 30 min. T3 in vitro (10(-6) M) increased the antioxidant activities of catalase, glutathione reductase (GR), GPx and glutathione level after 15/30 min, except SOD, substantiating in vivo effects in normal fish. The results suggest a rapid regulatory effect of thyroid hormone in vivo and in vitro, in the removal of reactive oxygen species in A testudineus.

Enzymatic characteristics of ligninperoxidases from Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus using n-propanol as substrate.

The activities of ligninperoxidases from Penicillium citrinum MTCC 3565, Fusarium oxysporum MTCC 3379 and Aspergillus terreus MTCC 3374 have been assayed and the enzymatic characteristics like Km, pH and temperature optima using n-propanol as the substrate have been reported. The results suggest that n-propanol can substitute veratryl alcohol as substrate for assaying ligninperoxidase activities from different fungal strains, without affecting the enzymatic characteristics. The above strains were selected, as they were known to secrete ligninperoxidase in the liquid culture medium.

Cysteine-179 of I kappa B kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

I kappa B kinase beta (IKK beta) subunit of IKK complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKK beta activation was reduced in the IKK beta (C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKK beta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKK beta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Extraçäo líquido-líquido de xilose redutase e xilitol desidrogenase por micelas reversas / Xylose reductase and xylitol dehydrogenase extraction liquid-liquid by reverse micelles

Células de Candida guilliermondii, obtidas de cultivos feitos en hidrolisado de bagaço de cana-de-açúcar, foram rompidas por sonicaçäo e os fragmentos celulares removidos por centrifugaçäo. As enzimas xilose redutase (XR) e xilitol desidrogenase (XD) foram separadas por extraçäo com micelas reversas, usando-se os tensoativos BDBAC, CTAB e AOT. A XR foi recuperada significativamente na fase aquosa de reextraçäo (FAII) em presença do BDBAC (fator de purificaçäo 4,8) ou CTAB (fator de purificaçäo 5,6). A XR, no entanto, ficou na fase aquosa de extraçäo (FAI) para ambos os tensoativos. Na presença do AOT näo se observou extraçäo significativa tanto da XR quanto da XD. Observou-se variaçäo desprezível nos valores...

Purificação de glicose-6-fosfato desidrogenase (G6PDH) em sistemas de duas fases aquosas utilizando PEG/citrato / Purification of glucose-6-phosphate dehydrogenase (G6PDH) by aqueous two-phase system by using PEG/citrate

Foi estudada, neste trabalho, a purificação por extração líquido-líquido da enzima glicose-6-fosfato desidrogenase (G6PDH), de Saccharomyces cerevisiae, em sistemas de duas fases aquosas, preparados com polietilenoglicol (PEG) e sal citrato. Foram avaliados, também, a estabilidade e o equilíbrio do sistema, além do efeito da concentração de citrato de sódio, concentração e massa molar do PEG na partição desta enzima, com auxílio de um planejamento experimental do tipo fatorial 2ü. Os resultados das extrações mostraram que é possível purificar a G6PDH nesse tipo de sistema, com a condução do processo em duas etapas. A primeira delas, conduzida com 17 porcento (p/p) de PEG 400 e 20 porcento (p/p) de citrato...

Purificação, caracterização, clonagem e seqüênciamento de beta-glicosidases de Tenebrio molitor (Coleoptera) / Beta-glycosidase of Tenebrio molitor (Coleoptera) purification, characterization, cloning and sequence

No lúmen do intestino médio da larva de Tenebrio molitor existem 4 "beta"-glicosidases (denominadas 1, 2, 3a e 3b), que não estão presentes na comida do animal. Elas foram purificadas usando-se técnicas de eletroforese e cromatografias de troca iônica e interação hidrofóbica. A "beta"-glicosidase 1 (Mr 59.000) é instável a 30ºC mas é estabilizada na presença do substrato. Ela praticamente não tem atividade sobre galactosídeos e cliva di- e oligossacarídeos. A enzima possui apenas 1 sítio ativo que apresenta 4 subsítios para ligação de glicose. Seu papel fisiológico deve ser o da clivagem de oligo- e principalmente dissacarídeos. A "beta"-glicosidase 2 (Mr 67.000) é muito instável a 30ºC e hidrolisar...

Lipasa hepática: efectos sobre el metabolismo lipídico y lipoproteico / Hepatic lipase: its effects on lipids and lipoproteins metabolism

La Lipasa Hepática (LH) es una enzima de síntesis y localización principalmente hepática. Como triglicérido hidrolasa y fosfolipasa interviene en la transformación de la lipoproteína de densidad intermedia (IDL) en lipoproteína de baja densidad (LDL) y en el catabolismo de la lipoproteína de alta densidad (HDL). Además actúa como ligando entre las lipoproteínas y distintos receptores de la superficie celular, en el catabolismo lipoproteico. En los últimos años, el estudio del polimorfisismo de su gen promotor (LIPC), ha esclarecido el comportamiento de la LH en diferentes situaciones metabólicas. Para la medida de su actividad, se validaron las condiciones finales del ensayo: sustrato de trioleína, sonicado en presencia de albúmina, en buffer Tris-HCl pH=9, con lisolecitina como emulgente, incubado en presencia de plasma post-heparínico. La actividad de la LH se encuentra bajo control hormonal; una disminución en su actividad genera un cuadro aterogénico, con hiperlipemia combinada y presencia de lipoproteínas aterogénicas. Por otro lado, un aumento marcado de LH, produce disminución de HDL y presencia de LDL pequeñas y densas. En este trabajo, se presentan los papeles lipolítico y no lipolítico de la LH, así como distintos factores que regulan su actividad y por lo tanto, su intervención en el proceso aterogénico

Activation of PKCdelta by tyrosine phosphorylation in rat parotid acinar cells

Receptor- and nonreceptor-mediated stimuli produce increases in both PKCdelta tyrosine phosphorylation and activity in rat parotid acinar cells and other cells. In vivo and in vitro increases and decreases in tyrosine phosphorylation resulted in increases and decreases, respectively, of PKCdelta activity. These studies demonstrated that increases in PKCdelta activity by G protein-coupled receptors and other stimuli were controlled by alterations in tyrosine phosphorylation.

An overview on chemical modification of enzymes. The use of group-specific reagents

Chemical modification is an important approach to the study of enzyme active sites. This article presents an overview of the methods used. The basic concepts, applications and limitations of chemical modification are outlined. The use of reagents specific for different amino acid side chains is also discussed

Chemical modification of enzymes: kinetic aspects [published erratum appears in Biol Res 1996; 29(2): 267]

Important information on enzyme ligand interactions can be obtained when analyzing the kinetics of chemical modification reactions. In this article, several kinetic models of inactivation are discussed, along with the determination of enzyme-ligand dissociation constants and of the pK of enzyme reactive groups from chemical modification kinetic data

Photoaffinity labeling and photoaffinity crosslinking of enzymes

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme

Chemical modification of allosteric properties of enzymes

Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase

Heterogeneity of late-onset adrenal 3ß-ol-hidroxysteroid dehydrogenase deficiency in patients with hirsutism and polycystic ovaries

Nine women clinical features of polycystic ovarian syndrome (PCOS) were studied in order to establish the differential diagnosis with late-onset adrenal hyperplasia (LOAH). Their hirsutism was classified as moderate in five patients and severe in the remainig four cases. All patients had bilateral polycystic ovarian enlargement by ultrasound examination. As a control group five women with normal ovarian function without hirsutism were submitted to the same protocol of study. The patients studied as well as the women of the control group had basal serum determinations of pregnenolone (P5), 17 hydroxypregnenolone (17-OHP5), dehydroepiandrosterone (DHEA), pregesterone (P), 17-hydroxyprogesterone (17-OHP), androstenedione (A), testosterone and cortisol by radioimmunoassay techniques. The basal serum levels of androgens showed no correlation with the severity of hirsutism or with the ultrasound finding. An adrenal stimulation with synthetic adrenocorticotropic hormone (ACTH) to all women performed in order to assess their adrenal responsiveness. The analysis of the ratios between delta 5 and delta 4 steroids demonstrated a partial enzymatic blockade at the level of 3ß-o1-hydroxysteroid dehydrogenase (3-HDS) in three patients. The blockade was particulary in the conversion of P5 to P and 17-OHP5 to 17- OHP. The lack of delta 4 steroid secretion in the presence of normal increase of delta 5 precursors following ACTH was noted. These findings confirm the clinical use of the ACTH stimulation test to reveal the presence of enzymatic alterations in adrenal steroidogenesis in some patients previously considered to have PCOS. Since it was demonstrated that the conversion steps were affected in variable degrees, the presence of different isoenzymes of 3-HSD is suggested

Efeito de diferentes níveis de fósforo sobre a atividade de fosfatases ácidas e a composição mineral de alguns fungos ectomicorrízicos / Effect of different levels of P no acid phosphatase activity and mineral composition of some ectomycorrhizal fungi

A atividade de fosfatases ácidas nos fungos ectomicorrízicos Rhizopogon reaii, Suillus sp, Pisolithus tinctorius 185, Pisolithus tinctorius 298 e Paxillus involutus aumentou com a diminuição dos níveis de P no meio de cultuvo. Em Rhisopogon nigrescens não se observou variação significativa na atividade desta enzima. A maior atividade fostatásica foi encontrada no fungo fP. involutus, a qual foi cerca de 16 vezes maior do que em P. tinctorius 185. A relação entre a atividade na ausência de P e na presença de 128 µM deste elemento (P0/P128) variou de 1.5 a 110 nos fungos R. nigrescens e P. involutus, respectivamente. EmP. tinctorius, a produção de matéria seca e os teores de P e de Ca com a produção de matéria seca, mas não com a atividade das fosfatases. Na ausência de P, o teor de Mg aumentou significativamente em relação àqueles em que o P estava presente, os quais não diferiram entre si. Além disso, o Mg apresentou alta correlação com a atividade fosfatásica, sugerindo um envolvimento de síntese protéica no processo de indução enzimática pela deficiência de P.

Cerebrospinal dehydrogenases in central nervous system infections.

Cerebrospinal fluid (CSF) dehydrogenases were studied in 42 controls, 23 children with pyogenic meningitis, 22 with tuberculous meningitis and 19 with encephalitis to assess their usefulness in differentiating between the different central nervous system infections. CSF-LDH and ICD activity was increased in CNS infections (p less than 0.0001), LDH being significantly higher (p less than 0.001) in pyogenic meningitis than in tuberculous meningitis or encephalitis. However, ICD activity was significantly different in each of these conditions (p less than 0.001). The dehydrogenase activity declined with subsequent clinical improvement, in all children with meningitis. A significant direct relationship was found between the enzyme activity and CSF protein content as well as total cell count. The 95% confidence interval confirms the utility of assaying CSF dehydrogenase activity to differentiate various CNS infections, thus improving the diagnostic ability.