Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Redox active macrocyclic receptors for neutral guests

A novel series of triazine-appended macrocyclic complexes has been investigated as potential hydrogen bonding receptors for complementarily disposed heterocycles. Cocrystallization of a melamine-appended azacyclam complex of Cull has been achieved with barbitone, the barbiturate anion and thymine. In each case, a complementary DAD/ADA hydrogen bonding motif between the melamine group and the heterocycle has been identified by X-ray crystallography. Electrochemical studies of the copper macrocycles in both nonaqueous and aqueous solution show anodic shifts of the CuII/I redox couple of more than 60 mV upon addition of guest molecules with matching H-bonding motifs. The Zn-II analogues have been synthesized via transmetalation of the Cull complex, and their guest binding properties investigated by NMR spectroscopy. H-1 NMR shifts of up to 0.8 ppm were observed upon addition of guest, and stability constants are similar to those obtained electrochemically.

Avaliação dos níveis séricos das vitaminas A, E, C e B2, de carotenóides e zinco, em idosos hospitalizados

Foram verificados os níveis séricos de zinco, carotenóides e vitaminas A, E, C, B2 em todos os idosos (n = 202) internados nas diversas enfermarias do hospital estudado, no período de fevereiro de 1986 a outubro de 1988. Foram estudados 130 homens e 72 mulheres que apresentaram média de idade de 67,8 anos, com variação entre 60 a 88. A percentagem de níveis séricos deficitários foi de 59,5 para o zinco, 56,5% para a vitamina C, 34,5% para a vitamina B2, 26% para a vitamina E, 13,2% para a vitamina A e 6,8% para os carotenóides. Os idosos portadores de leucoses, magaesôfogo, doença pulmonar obstrutiva crônica e insuficiência cardíaca congestiva constituíram-se no grupo de pacientes com grande prevalência de estado deficitário de zinco e das vitaminas estudadas, resultados que mostram a importância de se investigar as deficiências desses micronutrientes e dão subsídios para a abordagem terapêutica mais racional do paciente idoso internado.

Development of fire-resistant (B1 & B2) winter 1 KPU foams applied by gun or straw adapter and optimization of a gun applied winter 1 KPU foam

A realização do presente trabalho teve como principais objectivos o desenvolvimento de espumas de poliuretano de um componente com propriedades de resistência à chama superiores (B1 & B2), aplicadas por pistola ou por adaptador/tubo e a optimização de uma espuma de poliuretano de um componente de inverno aplicada por pistola. Todo o trabalho desenvolvido está dividido em dois projectos distintos: i. O primeiro projecto consistiu em desenvolver espumas de um componente com propriedades de resistência à chama (classificadas como B1 e B2 de acordo com a norma alemã DIN 4102), aplicadas por pistola (GWB1 e GWB2) ou por adaptador/tubo (AWB), utilizando polióis poliésteres aromáticos modificados e aditivos retardantes de chama halogenados. Estas espumas deveriam apresentar também propriedades aceitáveis a baixas temperaturas. Após realizar várias formulações foi possível desenvolver uma espuma AWB2 com apenas 3,3% de poliol poliéster no pré-polímero e com propriedades equivalentes às da melhor espuma comercial mesmo a 5/-10 (temperatura da lata/cura da espuma em °C) e também com uma altura de chama de apenas 11 cm. A partir de duas formulações (AWB2) que passaram o Teste B2, foram obtidas também, uma espuma GWB2 e outra GWB1 com propriedades equivalentes às da melhor espuma da concorrência a -10/-10 e a 23/5, respectivamente, embora não tenham sido submetidas ao teste B2 e B1 após as modificações efectuadas. ii. O segundo projecto consistiu em optimizar uma espuma de poliuretano de um componente de inverno aplicada por pistola (GWB3). A espuma inicial tinha problemas de glass bubbles quando esta era dispensada a partir de uma lata cheia, sendo necessário ultrapassar este problema. Este problema foi resolvido diminuindo a razão de GPL/DME através do aumento da percentagem em volume de DME no pré-polímero para 14% no entanto, a estabilidade dimensional piorou um pouco. O reagente FCA 400 foi removido da formulação anterior (6925) numa tentativa de diminuir o custo da espuma, obtendo-se uma espuma aceitável a 23/23 e a 5/5, com uma redução de 4% no custo da produção e com uma redução de 5,5% no custo por litro de espuma dispensada, quando comparada com a sua antecessora. Por último, foi avaliada a influência da concentração de diferentes surfactantes na formulação 6925, verificando-se o melhoramento da estrutura celular da espuma para concentrções mais elevadas de surfactante, sendo este efeito mais notório a temperaturas mais baixas (5/5). Dos surfactantes estudados, o B 8871 mostrou o melhor desempenho a 5/5 com a concentração mais baixa, sendo portanto o melhor surfactante, enquanto o Struksilon 8003 demonstrou ser o menos adequado para esta formulação específica, apresentando piores resultados globais. Pode-se ainda acrescentar que os surfactantes L-5351, L-5352 e B 8526 também não são adequados para esta formulação uma vez que as espumas resultantes apresentam cell collapse, especialmente a 5/5. No caso dos surfactantes L-5351 e L-5352, esta propriedade piora com concentrações mais elevadas. Em cada projecto foram também efectuados testes de benchmark em determinadas espumas comerciais com o principal objectivo de comparar todos os resultados das espumas desenvolvidas, em ambos os projectos, com espumas da concorrência.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0mVdecade−1 and 1.0×10−5 to 2.7×10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Cardiac beta-receptors in experimental Chagas' disease

Experimental Chagas' disease (45 to 90 days post-infection) showed serious cardiac alterations in the contractility and in the pharmacological response to beta adrenergic receptors in normal and T. cruzi infected mice (post-acute phase). Chagasic infection did not change the beta receptors density (78.591 ± 3.125 fmol/mg protein and 73.647 ± 2.194 fmol/mg protein for controls) but their affinity was significantly diminished (Kd = 7.299 ± 0.426 nM and Kd = 3.759 ± 0.212 nM for the control) p < 0.001. This results demonstrate that the alterations in pharmacological response previously reported in chagasic myocardium are related to a significantly less beta cardiac receptor affinity. During this experimental period serious cardiac cell alterations take place and functional consequences will be detected in the chronic phase.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay

Staphylococcus aureus binds Immunoglobulin G (IgG) on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN) against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA) with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01); but the former were killed more efficiently than the latter (80-90% and 40%, respectively). It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.

Expression of cyclooxygenase enzymes and prostaglandin E2 receptors in inflammatory airway diseases

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Functional and therapeutical implications of ligand recognition by the scavenger-like lymphocyte receptors CD5 and CD6

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect). Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.

Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni

INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER) and progesterone receptor (PR) expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR) showed statistically significant differences among the groups (p< 0.05), with significant low ER hormone expression in chronic infection, compared to control proliferative, control secretory and acute infection cases, and statistically significant high PR expression in both acute and chronic infection cases compared to the control secretory cases (p< 0.05). CONCLUSIONS: Schistosomiasis mansoni seems to have an important impact on the hormone expression of affected women. Further studies to explore the mechanism of such changes are recommended.