Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula)

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8 +/- 1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximate to 65 kDa, similar to that identified in other mammalian species, and binding of I-125-labelled human GH (hGH) was displaced by excess hGH (20 mug). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with I-125-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K-a) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96 +/- 0.2 x 10(9)/M, n = 6). Binding capacity (B-max) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4 +/- 0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol Chem 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed caves, using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

The blood volumes of the primary and secondary circulatory system in the Atlantic cod Gadus morhua L., using plasma bound Evans Blue and compartmental analysis

The volume of the primary (PCS) and secondary (SCS) circulatory system in the Atlantic cod Gadus morhua was determined using a modified dye dilution technique. Cod (N=10) were chronically cannulated in the second afferent branchial artery with PE-50 tubing. Evans Blue dye was bound to harvested fish plasma at a concentration of 1 mg dye ml(-1) plasma, and injected at a concentration of 1 mg kg(-1) body mass. Serial sampling from the cannula produced a dye dilution curve, which could be described by a double exponential decay equation. Curve analysis enabled the calculation of the primary circulatory and total distribution volume. The difference between these volumes is assumed to be the volume of the SCS. From the dilution curve, it was also possible to calculate flow rates between and within the systems. The results of these experiments suggest a plasma volume in the PCS of 3.42+/-0.89 ml 100 g(-1) body mass, and in the SCS of 1.68+/-0.35 ml 100 g(-1) body mass (mean +/- S.D.) or approximately 50% that of the PCS. Flow rates to the SCS were calculated as 2.7% of the resting cardiac output. There was an allometric relationship between body mass and blood volumes. Increasing condition factor showed a tendency towards smaller blood volumes of the PCS, expressed as percentage body mass, but this was not evident for the volume of the SCS.

Radio frequency plasma-induced hydrogen bonding on kaolinite

The radio frequency (RF) plasma-modified surfaces of kaolinite were investigated by diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and deuteration techniques to determine the nature of RF plasma-induced surface functional groups, the altered sites in the lattice, and interaction mechanism between RF plasma and the surface of the kaolinite. It has been concluded that the RF plasma-induced infrared (IR) vibration absorption bands at 2805, 3010, and 3100 cm(-1) are attributable to the stretching vibration of hydrogen-bonded hydroxyl groups, and the band at 1407 cm(-1) is attributable to the bending vibration of (HO-)Al-O or (HO-)Si-O groupings with hydrogen-bonded hydroxyl groups. Structural alteration occurred on both the surface and subsurface region of the kaolinite during RF plasma treatment. Further structural alteration or adjustment was also observed on well-modified and well-deuterated kaolinite. There are two types of OD bands visible in the DRIFT spectra of this kaolinite, one type which decreased rapidly as a function of time in moist air, and the other which remained unchanged during the measurement. Furthermore, the appearance of broad IR bands at 3500-3100 cm(-1) as a result of deuteration is evidence of structural disturbance by RF plasma treatment lattice deuteration. An RF plasma-induced hydrogen bonding model on the surface of the kaolinite is proposed.

Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

Plasma zinc and immune markers in runners in response to a moderate increase in training volume

Changes in plasma zinc concentration and markers of immune function were examined in a group of 10 male runners (n = 10) following a moderate increase in training over four weeks. Seven sedentary males acted as controls. Fasting blood samples were taken at rest, before (T0) and after T4) four weeks of increased (+ 16 %) training and after two weeks of reduced (- 31 %) training (W. Blood was analysed for plasma zinc concentration, differential leucocyte counts, lymphocyte subpopulations and lymphocyte proliferation using incorporation of H-3-thymidine. The runners increased their training volume by 16 % over the four weeks. When compared with the nonathletes, the runners had lower concentrations of plasma zinc (p = 0.012), CD3(+) (p = 0.042) and CD19(+) lymphocytes (p = 0.010) over the four weeks. Lymphocyte proliferation in response to Concanavalin A stimulation was greater in the runners (p = 0.0090). Plasma zinc concentration and immune markers remained constant during the study. Plasma zinc concentration correlated with total leucocyte counts in the athletes at T6 (r = -0.72, p < 0.05) and with Pokeweed mitogen stimulation in the nonathletes at T6 (r = -0.92, p < 0.05). Therefore, athletes are unlikely to benefit from zinc supplementation during periods of moderately increased training volume.

Desenvolvimento de metodologia para determinação de Co, Cr, Fe, Mn, Ni, Se e V em amostras de água produzida da indústria do petróleo através da técnica ICP OES utilizando digestão por micro-ondas

O descarte ou reutilização da água produzida da indústria do petróleo é difícil por causa dos impactos ambientais causados devido à presença de alta salinidade e componentes tóxicos, ou pelo risco de obstrução nas colunas de produção devido à formação de incrustações que causam redução na produção de petróleo e enormes perdas no processo de extração. Assim, o conhecimento da composição química da água produzida é muito importante. O método proposto por este trabalho visa a determinação de elementos traço (Co, Cr, Fe, Mn, Ni, Se e V) em amostras de água produzida de petróleo por espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES) utilizando a digestão ácida assistida por micro-ondas para o preparo das amostras (15 g de amostra e 2 mL de HNO3 concentrado). A curva analítica construída em HNO3 2% v v-1 foi adotada para o método após verificar que não é necessário o uso de salinidade para equiparação de matriz. Para o elemento Ni, não há necessidade do uso de padrão interno, para os elementos Co, Cr, Fe, Mn e V os melhores resultados foram obtidos usando Sc como padrão interno. Para o elemento Se é recomendado o uso de Y como padrão interno. Os limites de detecção obtidos foram Co 0,67, Cr 1,2, Fe 2,3, Mn 0,49, Ni 1,9, Se 3,7 e V 5,5 μg L-1; e os limites de quantificação foram Co 2,2, Cr 4,0, Fe 7,7, Mn 1,6, Ni 6,5, Se 12,4 e V 18,3 μg L-1. A exatidão do procedimento foi verificada através de testes de recuperação em dois níveis de concentração (40 e 80 μg L-1) e análise dos materiais certificados de referência de água estuarina SLEW-2 e de água do mar NASS-5. Bons valores de recuperação foram obtidos e não houve diferença significativa (95% de confiança) entre os resultados obtidos e os valores certificados dos materiais de referência.

Determinação de estanho em tecidos de ratas por ICP-MS

Os organoestânicos, principalmente o tributilestanho (TBT), são contaminantes ambientais, utilizados principalmente em tintas anti-incrustantes para navios. Eles sofrem bioacumulação e podem ser encontrados em mamíferos, inclusive em seres humanos. A principal fonte de exposição é a ingestão de alimentos contaminados. Esse estudo possuiu como objetivo a determinação de estanho em tecidos de ratas expostas cronicamente a TBT utilizando espectrometria de massas com plasma acoplado indutivamente (ICP-MS). Ratas Wistar adultas foram usadas na experimentação, sendo divididas dois grupos: o grupo exposto a 100 ng kg-1 dia-1 de TBT por 15 dias e o grupo de referência que recebeu somente o veículo durante o mesmo período de exposição. Ao final da exposição, os animais foram sacrificados e coletados plasma, coração, rim, pulmão, fígado e ovário para análise. As amostras foram secas em estufa por 72 horas e pulverizadas. A determinação de estanho foi realizada por ICP-MS após a digestão ácida assistida por micro-ondas de uma amostra com aproximadamente 100 mg. O limite de detecção (LD) calculado foi 4,3 ng L-1, o que permite a determinação de estanho em amostras de tecidos de animais usados para experimentação. A exatidão foi verificada pela análise do material de referência certificado de urina, Seronorm Urine (54,6 ± 2,7 μg L-1), tendo como resultado 50,1 ± 3,8 μg L-1. A concentração de estanho foi determinada em amostras de plasma, coração, rim, pulmão, fígado e ovário do grupo exposto a TBT e do grupo controle. Houve diferença estatisticamente significativa entre os dois grupos para todas as amostras analisadas. As diferenças entre os grupos foram mais pronunciadas nas amostras de fígado e rim. Além disso, este estudo mostrou que a presença de estanho no organismo de ratas distribui-se pelos tecidos acarretando em alterações morfofisiológicas já descritas em ovários, coração e fígado

Implementação e validação do método de determinação de metais e fósforo por ICP-OES (inductively coupled plasma-optical emission spectometry) em biodiesel

O objectivo do presente trabalho foi desenvolver, implementar e validar métodos de determinação de teor de cálcio (Ca), magnésio (Mg), sódio (Na), potássio (K) e fósforo (P) em biodiesel, por ICP-OES. Este método permitiu efectuar o controlo de qualidade do biodiesel, com a vantagem de proporcionar uma análise multi-elementar, reflectindo-se numa diminuição do tempo de análise. Uma vez que o biodiesel é uma das principais fontes de energia renovável e alternativa ao diesel convencional, este tipo de análises revela-se extremamente útil para a sua caracterização. De acordo com a análise quantitativa e qualitativa e após a validação dos respectivos ensaios, apresentam-se, na Tabela 1 as condições optimizadas para cada elemento em estudo. As condições de trabalho do ICP-OES foram escolhidas tendo em conta as características do elemento em estudo, o tipo de equipamento utilizado para a sua análise, e de modo a obter a melhor razão sinal/intensidade de fundo. Para a validação dos ensaios foram efectuados ensaios de recuperação, determinados limites de detecção e quantificação, ensaios de repetibilidade e reprodutibilidade, e verificação das curvas de calibração. Na tabela 2 apresentam-se os comprimentos de onda escolhidos (livres de interferências) e respectivos limites de detecção e quantificação dos elementos analisados por ICP-OES, na posição radial e radial atenuado.

Desilication of MOR zeolite: Conventional versus microwave assisted heating

MOR zeolites were modified via desilication treatments with NaOH, under conventional and microwave heating. The samples were characterized by powder X-ray diffraction, (27)Al and (29)Si NMR spectroscopy. TEM and N(2) adsorption at -196 degrees C. The acidity of the samples and the space available inside the pores were evaluated through a catalytic model reaction, the isomerization of m-xylene, for which the profiles of the coke thermal decomposition were also analyzed. Powder X-ray diffraction and (29)Si and (27)Al MNR results show that in comparison with conventional heating, microwave irradiation (a less time consuming process) leads to identical amount of Si extraction from the zeolite framework. With this treatment. in addition to the customary mesopores development promoted by conventional heating, a partial conversion of the zeolite microporosity into larger micropores, is observed. The microwave irradiated and conventionally heated samples show different catalytic behavior in the m-xylene isomerization model reaction. It was observed that, by controlling the experimental conditions, it is possible to obtain samples with catalytic properties closer to the parent material, which is also confirmed by the respective coke analysis. (C) 2011 Elsevier B.V. All rights reserved.