Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

Avaliação da aterosclerose subclínica e de níveis plasmáticos de LDL minimamente modificada em pacientes com espondilite anquilosante e sua correlação com a atividade da doença / Evaluation of sub-clinical atherosclerosis and plasma levels of minimally modified LDL in patients with ankylosing spondylitis and its correlation with disease activity

INTRODUÇÃO: A aterosclerose acelerada foi demonstrada em algumas doenças autoimunes, principalmente lúpus eritematoso sistêmico e artrite reumatóide. Embora a alta prevalência do uso de corticosteróides possa ser um fator complicador, por causa de seus efeitos prejudiciais em diversos fatores de risco, acredita-se que, nesses pacientes, a inflamação sistêmica per se desempenhe papel importante na aterogênese. MÉTODOS: Avaliamos a aterosclerose subclínica e os níveis plasmáticos de LDL eletronegativa circulante em pacientes com espondilite anquilosante (EA). Catorze pacientes que atendiam aos critérios de Nova York modificados para EA foram comparados com 13 controles equiparados. Avaliamos a espessura da íntima-média (EIM) na carótida por ultrassonografia bilateral da artéria carótida comum, artéria carótida interna e na bifurcação. Os grupos foram homogêneos, no que tange a fatores de risco cardiovasculares. Apenas um paciente no grupo de EA estava sendo medicado com corticosteróide. RESULTADOS: A presença de inflamação ativa foi demonstrada por BASDAI elevado e níveis mais elevados de PCR em pacientes versus controles (12,36 vs. 3,45 mg/dl, P=0,002). Não observamos diferença na EIM da carótida entre os dois grupos, em qualquer local da artéria. A média de EIM (6 mensurações em 3 locais pré-especificados, bilateralmente) foi 0,72 ± 0,28 no grupo de EA e 0,70 ± 0,45 mm nos controles (P=0,91). Também não observamos diferença significativa na LDL minimamente modificada entre pacientes e controles (14,03 ± 17,40 vs. 13,21 ± 10,21; P=0,88). CONCLUSÕES: Pacientes com EA não demonstraram aumento na EIM da carótida, em comparação com controles. Do mesmo modo, os níveis plasmáticos circulantes de LDL(-) não diferiram significativamente nos dois grupos.

INTRODUCTION: Accelerated atherosclerosis has been shown in some autoimmune diseases, mainly in Systemic Lupus Erythematosus and Rheumatoid Arthritis. Although high prevalence of corticosteroids use may be a confounding factor due to their detrimental effects on several risk factors, systemic inflammation per se is supposed to play an important role in atherogenesis in these patients. METHODS: We have evaluated sub-clinical atherosclerosis and plasma levels of circulating electronegative LDL, which represents the fraction of LDL that is minimally modified, in patients with ankylosing spondylitis (AS). Fourteen patients who fulfilled the modified New York criteria for AS were compared with 13 paired controls. Carotid intimal-media thickness (IMT) was assessed by ultrasonography bilaterally in common carotid artery, internal carotid artery and in the bifurcation. Groups were homogeneous regarding cardiovascular risk factors. Only a single patient in AS group was in use of corticosteroid. RESULTS: The presence of active inflammation was demonstrated by elevated BASDAI and higher CRP levels and in patients versus controls (12.36 vs. 3.45 mg/dl, P = 0.002). No difference was found in carotid IMT between both groups, in any site of artery. Averaged IMT (6 measurements, at 3 pre-specified sites bilaterally) was 0.72 ± 0.28 in AS group and 0.70 ± 0.45 mm in controls (P = 0.91). Minimally modified LDL did not differ significantly either between patients and controls (14.03 ± 17.40 vs. 13.21 ± 10.21; P = 0.88). CONCLUSIONS: Patients with AS did not show increased carotid IMT in comparison to controls. In the same way, circulating plasma levels of LDL (-), did not differ significantly in both groups.

Efecto del propóleos chileno sobre el metabolismo de glucosa en ratones diabéticos / Effect of chilean propolis on glucose metabolism in diabetic mice

En el presente estudio se evaluó el efecto del propóleos sobre el metabolismo de la glucosa en ratones C57/BL-6 con diabetes mellitus tipo 2 inducida por dieta alta en grasa. Se midieron los cambios en las concentraciones séricas de lípidos, glucosa e insulina, y el efecto sobre la captación de 2-deoxi-[2,6-3H]-D-glucosa, síntesis de [14C]-glicógeno y descarboxilación de [U-14C]-D-glucosa inducida por insulina en músculo aislado. Los resultados muestran que en ratones diabéticos, el tratamiento con propóleos (150 mg/kg/día) reduce los niveles de insulina e índice HOMA (P<0.05). También disminuyó la obesidad abdominal de estos animales (P<0.05). Por otro lado, no modificó las concentraciones plasmáticas de glucosa, colesterol total y triglicéridos. Se observó también que la captación de 2-deoxi-[2,6-3H]-D-glucosa, síntesis de [14C]-glicógeno y descarboxilación de [U-14C]-D-glucosa inducida por insulina en músculo sóleo de ratones tratados con propóleos fue significativamente superior al grupo control (P<0.05). En resumen, nuestros datos confirman que el propóleos es capaz de modular el metabolismo de glucosa en ratones C57/BL-6 con diabetes mellitus tipo 2 inducida por dieta alta en grasa. Los datos obtenidos constituyen un importante antecedente que avala el posible uso del propóleos como fuente de polifenoles con actividad antidiabetogénica.

In the current study, we investigated the effect of propolis on diabetic mice undergoing propolis treatment (150 mg/kg/day) for a 6 week period. We also evaluated serum lipids, glucose, insulin levels and the effect on glucose uptake of 2-deoxy-D-[2,6-3H] glucose, [14C]-glycogen synthesis and [U-14C]-D-glucose decarboxylation induced by insulin in muscle tissue. Our results show that treatment with propolis (150 mg/kg/day) reduced insulin and HOMA index (P<0.05). Propolis also lowered abdominal obesity (P<0.05). No effects over serum glucose, total cholesterol and triglycerides levels were observed. We also observed that uptake of 2-deoxy-D-[2,6-3H] glucose, [14C]-glycogen synthesis and [U-14C]-D-glucose decarboxylation induced by insulin in soleus muscle of mice treated with propolis were significantly greater than control group (P<0.05). In summary, our data establishes that propolis modulates glucose metabolism. This result constitutes important data indicating that propolis can be used as a polyphenols source with antidiabetogenic activity.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

LDL: da síndrome metabólica à instabilização da placa aterosclerótica / LDL: from metabolic syndrome to instability of the atherosclerotic plaque

A dislipidemia da síndrome metabólica (SM) confere elevado risco cardiovascular e caracteriza-se por aumento dos triglicérides, diminuição da HDL e alterações qualitativas da LDL, tornando-a mais aterogênica, como a LDL pequena e densa. LDLs modificadas (LDLm) foram detectadas in vivo no plasma e em placas ateroscleróticas. Uma pequena porcentagem do total de LDLs plasmáticas apresenta maior carga negativa na superfície [LDL(-)], sendo uma sub-população heterogênea de partículas com maior poder de agressão ao endotélio. Origina-se da oxidação, glicação ou outros processos que alteram sua composição química, estando aumentada em indivíduos diabéticos, hipercolesterolêmicos e naqueles com doença isquêmica cardíaca. A LDLm, ao ser fagocitada pelo receptor scavenger do macrófago, transforma-o numa célula espumosa e inicia uma reação imune-inflamatória. A participação da LDLm no processo aterogênico continua até a ruptura da placa e trombogênese, quando ela induz apoptose em células endoteliais e musculares lisas, aumenta a produção de metaloproteinases que digerem a matriz, fragilizando a cápsula, e exacerba a inflamação que concorre para o desenvolvimento do trombo. O aprimoramento dos ensaios laboratoriais para a LDLm permitirá maior aplicabilidade clínica, melhorando o poder preditivo de eventos cardiovasculares em relação ao perfil lipídico convencional e demais fatores de risco presentes na SM.

The dyslipidemia of the metabolic syndrome (MS) confers an elevated cardiovascular risk and is characterized by increased concentrations of triglycerides, decreased HDL-cholesterol and qualitative alterations in LDL which renders it more atherogenic, like the small dense LDL. Modified forms of LDL (mLDL) have been detected in vivo in the plasma and atherosclerotic plaques. A minor fraction of the total LDL has an electronegative charge and is represented by a heterogenic subpopulation of particles [LDL(-)], with higher potential to induce endothelial injury. It could be derived from oxidation, glication or other processes that alter its chemical composition and is increased in diabetic, hypercholesterolemic subjects, and in those with established coronary artery disease. mLDL are internalized by macrophages through scavenger receptors, originating foam cells and inducing an immune-inflammatory reaction. In the atherosclerotic process, the action of mLDL continues until plaque rupture and thrombogenesis, when it promotes apoptosis in endothelial and smooth muscle cells, and activates matrix metalloproteinases, weaken the fibrous cap, and further enhance the inflammatory process that ends in the thrombus formation. Development of new laboratory methods is necessary to enhance the clinical applicability of mLDL and the predictive power of the conventional lipid profile and other cardiovascular risk factors of the MS.

Alcaptonúria: relato de dois casos e revisão bibliográfica / Alcaptonuria: A propos of two cases and bibliographic review

A alcaptonúria é uma doença rara, na qual ocorre a formaçäo de um pigmento que se acumula na cartilagem, pele e tecidos conectivos (ocronose), resultando em artrite, lesöes cardiovasculares, hiperpigmentaçäo da pele e outras patologias. Os autores descrevem dois casos de alcaptonúria, detectados em dois irmäos, sendo um do sexo masculino, com idade de 12 anos, e outro do sexo feminino, com dois meses de idade, que foram diagnosticados através da anamnese e dosagem de AH urinário por cromatografia.

Prevalence of sorologic markers of HBV and HCV infection in HIV-1 seropositivr patients in Florionópolis, Brazil

B and C hepatitis are infections transmitted in ways similar to the human immunodeficiency virus (HIV). The present study was designed to assess the prevalence of hepatitis B (HBV) and hepatitis C (HCV) virus infection in HIV-1-infected patients who lived in the metropolitan areas of Florianópolis, in the State of Santa Catarina. Ninety-three patients seropositive to HIV-1 were identified by using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence. Patients were assigned to according to HIV-1 transmission risk: homosexuals (n=20), heterosexuals (n=39), and intravenous drugs users (IVDU) (N=34). HbsAg, Anti-HBc, Anti-HBc IgM and Anti-HCV measurements were done using ELISA. HBV and HCV prevalence in HIV-1 infected patients was 71 percen and 53.8 percent, respectively. In the IVDU group, the prevalence of HBV (85.3 percent), of HCV (88.2 percent), and infections by both viruses (76.5 percent) was greater than that observed in the heterosexual and homosexual groups. Hepatitis B markers (HbsAg and Anti-HBc) for chronic persistent disease were recorded in 24.3 percent of patients, and for past infection in 71.2 of patients. The evidence of high prevalence of HBV and HCV infections in HIV-1 seropositive patients, mainly among intravenous drug users, should be included in educational programs in an effort to decrease the incidence of multiple infections.

Lipoprotein oxidation: implications to atherogenesis

Lipoprotein modification is a critical step in the development of atherosclerosis. Oxidant species released by endothelial cells, smooth muscle cells, monocytes, neutrophils, macrophages and platelets can oxidatively modify lipoproteins. Oxidized lipoproteins generated in vivo may participate in the atherogenic process by different mechanisms. This review will focus on the processes leading to lipoprotein oxidation, the interactions of oxidized lipoproteins with vascular endothelium and their participation in atheroma formation, the disturbances induced by lipoprotein oxidative modifications on intravascular lipoprotein metabolism, and the occurrence of oxidative stress in subjects with hyperlipoproteinemia. The elucidation of these events may be important to establish future preventive strategies and therapeutic approaches for atherosclerosis.

Enolizable carbonyl and imino metabolites may act as endogenous sources of reactive oxygen species

Highly reactive oxyradicals and electronically excited triplet carbonyls can be generated in vitro by iron complexes and heme enzyme-catalyzed aerobic oxidation of synthetic or naturally occurring substances capable of enolization in aqueous medium. Monoenols and enamines, obtained by (alpha-methyne-carbonyl and -imine enolization, undergo dioxygen insertion and ultimately originate triplet species; e.g., isobutanal, 3-methylacetoacetone, Schiff bases. In turn, (alpha-hydroxy- and (alpha-aminocarbonyls (e.g., carbohydrates, 5-aminolevulinic acid) tautomerize to enediols and enolamines and yield oxyradicals, initiated by electron transfer to dioxygen, as polyphenols (e.g., 6-hydroxydopamine) and polyphenolamines do. Free radicals and excited species have been implicated in several normal and pathological processes. We here briefly review our contributions to this research area, emphasizing a possible in vivo prooxidant role for 5-aminolevulinic acid, the heme precursor accumulated in several porphyric disorders (e.g., lead poisoning, acut intermittent porphyria, tyrosinosis).

Avaliação das enzimas séricas em pacientes com tumores malignos / Enzime evalluation in serum of patients with malignancies

Foram determinadas as atividades da lactato desidrogenase (LD), creatina-quinase CK), aspartato aminotransferase (AST), alanina aminotransferase (ALT), gama-glutami transpeptidase (gama-GT) e fosfatase alcalina (ALP), bem como das isoenzimas da LD e da fração B da CK (CK-B), no soro de indivíduos portadores de tumores malignos primários em vários órgãos. Entre as enzimas estudadas, a relação CK-B/CK-Total (%CK-B), foi o parâmetro mais sensível na detecção desses processos neoplásticos, principalmente como indicador da presença de metástases. As isoenzimas LD4 e LD5 revelam-se mais específicas que a LD total na detecção do câncer. Gama-GT e ALP foram as enzimas mais indicativas de comprometimento hepático de origem metastática.