B7-H3 and CD 133 expression in non-small cell lung cancer and correlation with clinicopathologic factors and prognosis

To detect the expression of B7-H3 and CD 133 in human non-small cell lung cancer [NSCLC] specimens and lung benign lesions, and to evaluate the correlation between the 2 biomarkers and clinicopathologic features. This is a case-control study of 102 tissue specimens collected from NSCLC participants undergoing thoracic surgery in the Second Affiliated Hospital of Soochow University, Suzhou, China, between January 2006 and December 2008. From the 102 patients, 25 adjacent non-cancer samples were verified pathologically as normal tissue [positive group], and 24 benign inflammatory lesion tissues were used as control [negative group]. Specimens from 126 participants were stained immunohistochemically using Image-Pro Plus software, and the cell number was measured in each section. Of the 102 specimens, 71 expressed B7-H3, and 51 expressed CD 133, higher than that in benign lesions [p<0.001] or non-cancer tissues [p<0.001]. B7-H3 expression in squamous cell carcinoma [SCC] was significantly higher than those in adenocarcinoma [p=0.048], while CD 133 expression in large cell lung carcinoma was higher than that in SCC [p=0.023]. The mean number of tumor-infiltrating lymphocytes [TILs] in the B7-H3-positive group was lower than that in the B7-H3-negative group [p=0.026]. The mean TILs in the CD133-positive group was significantly lower than that in CD133-negative group [p=0.029]. We found that CD 133 was related to tumor cell differentiation degree and CD 133 expression was negatively correlated with B7-H3 expression. The CD 133 positive or B7-H3 negative was associated with poor prognosis of NSCLC patients by Cox regression analysis. Both CD 133 and B7-H3 might induce apoptosis of TILs in NSCLC and tumor evading host immune surveillance. Either CD 133 or B7-H3 might be an independent risk factor of NSCLC participants

Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-gamma and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.

Factors affecting the expression of recombinant glycoproteins.

N-glycosylation is both species and tissue specific with a series of membrane bound glycosidases and glycosyltransferases modifying the oligosaccharide as it moves through the endoplasmic reticulum (ER) and Golgi. Each of these individual enzymatic reactions may not go to completion; therefore giving rise to glycoforms of the polypeptide. Glycosylation patterns of recombinant proteins are relevant for the immunogenicity, the pharmacological activity, pharmacokinetic profile, solubility and stability of the protein. This review describes the effect of primary and the 3-dimensional structure of the protein on sequon occupancy. Heterogeneity due to cell specific glycosylation and tissue culture conditions are discussed with main emphasis on N-glycosylation sequon occupancy. The review also discusses how fully glycosylated with total sequon occupancy glycoproteins which are of prime relevance in the expression of pharmaceutically relevant glycoproteins can be obtained.

Expresión de azúcares endometriales y del oviducto durante el ciclo reproductivo de la coneja / Expression of endometrial and oviductal glycoproteins during the reproductive cycle of female rabbit

Antecedentes: Numerosos factores han sido implicados en el proceso de implantación, entre ellos se destaca la presencia de glicoproteínas de superficie. Se ha demostrado en ratones, entre otros, que los tipos y cantidades de azúcares de superficie varían según el período del ciclo reproductivo en el que se encuentran, lo que podría sugerir un importante papel de estas variaciones en la mayor receptividad del útero al blastocisto. Objetivos: Describimos los carbohidratos de superficie del aparato reproductor de la coneja, correlacionándolos con los cambios en sus genitales externos, y con los distintos períodos de su ciclo reproductivo. Métodos: Se usaron 15 conejas, ovuladoras coitales, en las que se identifica el período del ciclo según cambios en sus genitales externos; 5 tenían vulva blanca (poca receptividad al macho y bajo índice de embarazos), 5 tenían vulva rosa, y 5 roja (máxima receptividad al macho y alto índice de embarazos). Se estudiaron variaciones en los azúcares de superficie en el oviducto, útero distal, útero medio y cuello uterino, representados por el grado de tinción de 5 lectinas: eritrina cristagalli (ECL), dolichos biforus agglutinin (DBA), ulex europaeus aglutinin-1(UEA-1), pisum sativum agglutinin (PSA) y artocarpus integrifolia (Jacalin). Resultados: Hubo predominio del disacárido N-acetilgalactosamina alfa 1-3N-acetilgalac-tosamina (DBA) en todos los períodos y lugares del aparato reproductor, exceptuando el período de vulva roja en útero medio, donde prevalece la beta galactosa (Jacalina). Los residuos de manosa/alfa glucosa (PSA), presentan alta reacción en los períodos de vulva rosa y roja en el oviducto y útero distal. La galactosa beta 1-4 N-acetil glucosamina (ECL) y la L-fucosa (UEA-1) tienen bajo nivel de expresión en todos los sitios y períodos. Conclusión: La ausencia del disacárido N-acetilgalactosamina alfa 1-3N-acetilgalac-tosamina en el período de vulva roja y en el útero medio y la prevalencia de la beta galactosa en...

La proteina asociada a SLAM (SAP) regula la expresion de IFN-gamma en lepra / The SLAM-associated protein (SAP) regulates IFN-gamma expression in leprosy

La inmunidad protectora contra Mycobacterium leprae requiere IFN-gamma. Los pacientes con lepra tuberculoide producen localmente citoquinas Th1, mientras que los pacientes lepromatosos producen citoquinas Th2. La molécula linfocitaria activadora de señales (SLAM) y la proteína aociada a SLAM (SAP) participan en la diferenciación celular que conduce a producción de patrones específicos de citoquinas. A fin de investigar la vía SLAM/SAP en la infección por M. leprae, determinamos expresión de ARN mensajero (ARNm) de SAP, IFN-gamma y SLAM en pacientes con lepra. Obeservamos que la expresión de SLAM correlacionó en forma directa con la expresión de IFN-gamma, mientras que la expresión de SAP interferiría con las respuetas de citoquinas Th1 mientras que SLAM contribuiría con la respuesta Th1 en lepra, señalando a la vía SLAM/SAP como potencial blanco modulador de citoquinas en enfermedades con respuestas Th2 disfuncionales.

High extracellular Ca2+ alone stimulates osteoclast formation but inhibits in the presence of other osteoclastogenic factors

High ambient Ca2+ at bone resorption sites have been implicated to play an important role in the regulation of bone remodeling. The present study was performed to clarify the mode of high extracellular Ca2+ (Ca2+e)-induced modulation of osteoclastogenesis and the expression of receptor activator of nuclear factor-kB ligand (RANKL) and osteoprotegerin (OPG), thereby to define its role in osteoclast formation. Mouse bone marrow cells were cocultured with osteoblastic cells in the absence or presence of osteoclastogenic factors such as 1,25-dihydroxyvitaminD3 (1,25-(OH)2vitD3) and macrophage colony-stimulating factor/soluble RANKL. Ca2+ concentration in media (1.8 mM) was adjusted to 3, 5, 7 or 10 mM. Osteoclast formation was confirmed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and the expression of osteoclast phenotypic markers (calcitonin receptor, vitronectin receptor, cathepsin K, matrix metalloproteinase-9, carbonic anhydrase 2). High Ca2+e alone significantly stimulated osteoclast formation in a dose-dependent manner. However, in the presence of highly osteoclastogenic factors, high Ca2+e significantly inhibited osteoclastogenesis. High Ca2+e alone continuously up-regulated RANKL expression while only transiently increased OPG expression. However, in the presence of 1,25-(OH)2vitD3, high Ca2+e did not change the 1,25-(OH)2vitD3- induced RANKL expression while increased OPG expression. Taken together, these findings suggest that high Ca2+e alone increase osteoclastogenesis but inhibit in the presence of other osteoclastogenic factors. In addition, high Ca2+e-induced osteoclastogenesis may be mediated by osteoblasts via up-regulation of RANKL expression. Meanwhile up-regulated OPG might participate in the inhibitory effect of high Ca2+e on 1,25-(OH)2vitD3-induced osteoclastogenesis.

Expression of Osteoprotegerin and RANK Ligand in Breast Cancer Bone Metastasis

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.

Infección in vitro por diferentes cepas de Paracoccidioides brasiliensis / In vitro infection by different strains of Paracoccidioides brasiliensis

We analyzed the in vitro infection process by P. brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines. The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent). Strains of intermediate and high virulence had higher adherence at first contact (about 16). Strain B339 had a slower adherence at first contact (8) than the others during the same period. The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains. We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro. We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

Leukocyte protein-bound carbohydrate components in diabetes mellitus

The protein bound components [hexose, hexosamine and sialic acid] in the leukocytes of type I type II diabetic patients and age and sex matched non-diabetic subjects were determined. The mean values of plasma glucose and glycated hemoglobin of type I patients were 201 +/- 39 mg/d1 and 11.7 +/- 2.48% respectively. The repective values of type II patients were 176 +/- 39 mg/dl and 11.34 +/- 2.1%. The levels of hexose, hexosamine and sialic acid were significantly higher in diabetic patients than those of age and sex matched non-diabetic control subjects. The increased protein bound carbohydrate components may cause an increase in the levels of glycoprotein material which might result in the thickening of the basement membranes characteristic of diabetes mellitus

Actualidades sobre bioquímica de la vitamina A / Biochemistry of vitmin A: a contribution

Con el propósito de investigar los compuestos presentes en el hígado derivados del metabolismo, se inocularon intraperitonealmente ratas hembras de 8 semanas de nacida con (11,12 H)- acetato de retinol en concentraciones fisiológicas. Por técnicas cromatográficas se demostró la presencia de tres compuestos radioactivos, dos con características no polar (I y II) y un tercero polar (III). El compuesto I se identificó como palmitato de retinol, lo cual permite concluir que solamente se almacena en el hígado bajo esa forma. Utilizando técnicas de doble marcaje isotópico, con (3Hz)-retinol y (14 C)-galactosa, ó (3H9-retinol y (32P)-fosfato, se demostró en el compuesto III la presencia de los tres isótopos. Por sus propiedades de hidrólisis ácida y alcalina en condiciones suaves, se concluyó que el derivado polar III es fosfato de retinil galactosa. Este compuesto podría ser el donador de restos galactosil en biosíntesis de glicoproteínas

Conserved structural features in glycoprotein processing glucosidase I from several tissues and species.

Glucosidase I initiates the processing of the oligosaccharide, Glc3Man9GlcNAc2, in newly assembled glycoproteins by excising the distal alpha 1,2-linked glucosyl residue in the oligosaccharide. Earlier, the enzyme purified from the ER of rat and bovine mammary gland has been found to have M(r) of 85 kDa, as examined by SDS-PAGE along with a domain structure in which a 39 kDa lumenally-oriented region is anchored to the ER through a transmembrane segment and a short cytoplasmic tail. These studies were further extended to include the enzyme from several different tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. Using anti-rat glucosidase I antibody as a probe and several biochemical parameters such as SDS-PAGE analysis, trypsin-catalyzed digestion, ConA-binding, endo H susceptibility and peptide mapping analysis by cleavage of the tryptophanyl peptide linkages within the enzyme, it was found that glucosidase I in all of the tissue sources examined has an M(r) of 85 kDa and is cross-reactive to anti-rat glucosidase antibody. The enzyme is a high mannose glycoprotein, and has domain features in its structure; the enzyme from mouse, rat, guinea pig and bovine mammary glands and sheep liver is sequentially cleaved by trypsin to generate fragments of 69, 55 and 39 kDa. The rate of release of the different fragments differs for different sources, indicating some evolutionary changes in its primary structure. The trypsin-released fragments from pig kidney enzyme are 69, 45 and 29 kDa in size, identical to the same observed earlier for pig liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinación colorimétrica de proteínas glicadas en sangre entera. Recolección de muestras en papel de filtro. Valores de referencia en niños / Colorimetric determination of glycated whole blood protein collection of samples on filter paper. Reference values on children

Las proteínas glicadas en sangre entera (PGSE) constituyen un parámetro que permite el seguimiento del control glucémico en pacientes con Diabetes mellitus, ofreciendo información de la glucemia promedio en los dos a tres meses previos. En el presente trabajo se llevó a cabo la puesta a punto del método colorimétrico del ácido 2-tiobarbitúrico (ATB) para medir PGSE, recolectada en papel de filtro. El fundamento del método consiste en la conversión del glúcido unido a la proteína a 5-hidroximetilfurfural (5-HMF) por calentamiento a 100 C en presencia de ácido oxálico. Por reacción entre 5-HMF y ATB se forma un compuesto coloreado que puede cuantificarse a 443 nm. Se estableció una comparación de los resultados de PGSE con HbA, medida por cromatografía de intercambio iónico (r=0,505) y por método colorimétrico del ATB (r=o,948). Este último estudio comparativo permite demostrar que las PGSE están constituidas en un 70-90% por HbA1, correspondiendo el porcentaje restante a proteínas séricas glicadas. También se estudió una población infantil normal obteniéndose un intervalo de referencia para PGSE de 41-87 nmol fructuosa/10mg proteínas; y una población infantil diabética, tipo I, observándose que el método en estudio permite diferenciar claramente los pacientes diabéticos compensados de aquellos no compensados. Los C.V.% intra e interensayo para estándares fueron inferiores a 4,3 y 5,3% respectivamente, en tanto para muestras fueron de 4,5 y 6,5% respectivamente. Además se observó que el método es sensible, barato y presenta ventajas respecto de otros métodos, ya que elimina la interferencia de hemoglobinopatías, aductos de Hb no glicadas y forma aldimina inestable; y no es influido por el pH y la temperatura

Studies on hormonal modulation of asparagine-linked glycoprotein biosynthesis in explant cultures of rat mammary gland.

Glucosidase I has been purified to homogeneity and polyclonal antibodies against the enzyme have been prepared. The anti-glucosidase I antibodies recognized a single band of 85 kDa on western blot at a dilution as high as 1:2000 and also inhibited the enzyme activity, suggesting the specificity of the antibodies. Con A-Sepharose binding experiment indicates that this enzyme itself is a high mannose type N-linked glycoprotein. The increase in the electrophoretic mobility of 85 kDa band following digestion with endoglycosidase H and F strengthened this observation. The presence of any O-linked sugar attached covalently to glucosidase I could not be detected by binding assays with O-linkage specific biotinylated lectins. The studies on developmental regulation suggest that the synthesis of glucosidase I is modulated with the ontogeny of the gland. Lactogenic hormones, viz. insulin, hydrocortisone and prolactin, appeared to regulate the synthesis of glucosidase I. The possible role of these hormones in the overall regulation of protein N-glycosylation has been discussed.