Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

Avaliação de fatores virológicos associados ao desenvolvimento de carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica

O objetivo principal deste estudo foi avaliar fatores virais associados com a evolução para o carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica. Para tanto caracterizamos os subgenótipos do HBV, investigamos a ocorrência de mutações nos genes pré-core/core do HBV associadas à presença de CHC avaliamos por análise filogenética a associação de linhagens virais com a ocorrência de CHC e por fim a associação de outros fatores de risco com o desenvolvimento de CHC. Foram incluídos 119 amostras de soro de pacientes com infecção crônica pelo HBV, destas amostras 60 pertencem ao grupo 1 (CHC), que são pacientes com diagnóstico confirmado de carcinoma hepatocelular e 59 amostras pertencem ao grupo 2 (sem CHC) que são pacientes com hepatite crônica sem detecção prévia de nódulos hepáticos. Foram obtidas informações acerca da idade, sexo e naturalidade. Além disso, os pacientes responderam a um questionário sobre fatores de riscos associados ao desenvolvimento de CHC. Foram realizados exames bioquímicos, sorológicos, determinação da carga viral, e amplificação por nested PCR e sequenciamento das regiões S/polimerase e pré-core/core do genoma viral para posterior caracterização dos genótipos/subgenótipos do HBV e pesquisa de mutações associadas com evolução da doença hepática. Em relação à idade e sexo não houve grande variação entre os grupos. Quanto à naturalidade a maioria era procedente da região sudeste, seguido pela região nordeste; e por fim seis pacientes eram procedentes de outros países. Com base no sobrenome dos pacientes avaliou-se também a frequência de etnia oriental na casuística estudada, que foi similar nos 2 grupos. O perfil sorológico HBeAg negativo foi o mais frequente nos dois grupos de pacientes, assim como níveis de carga viral abaixo de 2.000 UI/mL. Em relação aos exames bioquímicos foram observadas diferenças estatisticamente significantes nos níveis séricos de AFP (p= 0,0013), FA (p= 0,0003) e GGT (p= 0,005). Dentre os fatores de risco analisados neste estudo, o consumo de amendoim foi o único que apresentou significância estatística (p= 0,003). A região S/pol foi amplificada e sequenciada com sucesso em 58 amostras (28 do grupo 1 e 30 do grupo 2). Entre as 58 amostras analisadas 4 genótipos e 8 subgenótipos do HBV foram identificados, sendo o subgenótipo A1 o mais frequente nos dois grupos. Não se observou diferença estatisticamente significante na distribuição dos subgenótipos entre os dois grupos de pacientes. Na topologia da árvore filogenética construída com sequências do HBV isoladas dos pacientes incluídos neste estudo e sequências disponíveis no GenBank não se observou padrões de agrupamento associados com o perfil clinico do paciente (com e sem CHC). Foram obtidas sequências de boa qualidade da região précore/ core em 44 amostras, sendo 20 amostras do grupo 1 e 24 do grupo 2. Diversas das mutações investigadas foram identificadas na região précore/ core, as quais foram avaliadas estatisticamente para verificar a existência de diferença na frequência das mesmas entre os grupos de pacientes estudados. Entre as mutações identificadas se destacaram com significância estatística as seguintes mutações: T1768A (p= 0,006), a combinação das mutações C1766T + T1768A (p= 0,043) e G1888H (p= 0,05). Na análise de regressão logística simples foi possível identificar que a chance de um paciente do grupo 2 desenvolver CHC aumenta 14,7 vezes na presença de infecção por cepas do HBV com a mutação T1768A, enquanto que a infecção com cepas do HBV que albergam a mutação G1888H reduz tal chance 2,5 vezes

Effects of nitrogen supply on xylem cytokinin delivery, transpiration and leaf expansion of pea genotypes differing in xylem-cytokinin concentration

The rms2 and rms4 pea ( Pisum sativum L.) branching mutants have higher and lower xylem-cytokinin concentration, respectively, relative to wild type (WT) plants. These genotypes were grown at two levels of nitrogen (N) supply for 18 - 20 d to determine whether or not xylem-cytokinin concentration (X-CK) or delivery altered the transpiration and leaf growth responses to N deprivation. Xylem sap was collected by pressurising de-topped root systems. As sap-flow rate increased, X-CK declined in WT and rms2, but did not change in rms4. When grown at 5.0 mM N, X-CKs of rms2 and rms4 were 36% higher and 6-fold lower, respectively, than WT at sap-flow rates equivalent to whole-plant transpiration. Photoperiod cytokinin (CK) delivery rates ( the product of transpiration and X-CK) decreased more than 6-fold in rms4. Growth of plants at 0.5 mM N had negligible (< 10%) effects on transpiration rates expressed on a leaf area basis in WT and rms4, but decreased transpiration rates of rms2. The low-N treatment decreased leaf expansion by 20 - 25% and expanding leaflet N concentration by 15%. These changes were similar in all genotypes. At sap-flow rates equivalent to whole-plant transpiration, the low N treatment decreased X-CK in rms2 but had no discernible effect in WT and rms4. Since the low N treatment decreased transpiration of all genotypes, photoperiod CK delivery rates also decreased in all genotypes. The similar leaf growth response of all genotypes to N deprivation despite differences in both absolute and relative X-CKs and deliveries suggests that shoot N status is more important in regulating leaf expansion than xylem-supplied cytokinins. The decreased X-CK and transpiration rate of rms2 following N deprivation suggests that changes in xylem-supplied CKs may modify water use.

Yield response of rice (Oryza sativa L.) genotypes to drought under rainfed lowlands 4. Vegetative stage screening in the dry season

Screening for drought resistance of rainfed lowland rice using drought score (leaf death) as a selection index has a long history of use in breeding programs. Genotypic variation for drought score during the vegetative stage in two dry season screens was examined among 128 recombinant inbred lines from four biparental crosses. The genotypic variation detected for drought score in the dry season was used to examine the reliability of the dry season screening method to estimate relative grain yield of genotypes under different types of drought stress in the wet season. Large genotypic variation for drought score existed in two experiments (A and B). However, there was no relationship between the drought scores of genotypes determined in these two experiments. Different patterns of development and severity of drought stress in these two experiments, i.e. slow development and mild plant water deficit in experiment A and fast development and severe plant water deficit in experiment B, were identified as the major factors contributing to the genotypes responding differently. Larger drought score in the dry season experiments was associated with lower grain yield under specific drought stress conditions in the wet season, but the association was weak to moderate and significant only in particular drought conditions. In most cases, a significant phenotypic and moderate genetic correlation between drought score in the dry season and grain yield in the wet season existed only when both drought score and grain yield of genotypes were affected by similar patterns and severity of drought stress in their respective experimental environments. The dry season environments used to measure genotypic variation for drought score should be managed to correspond to relevant types of drought environment that are frequent in the wet season. The efficiency of using the drought score as an indirect selection criterion for improving grain yield for drought conditions was lower than the direct selection for grain yield, and hence wet season screening with grain yield as a selection criterion would be more efficient. However, using drought score as a selection index, a larger number of genotypes can be evaluated than for wet season grain yield. Therefore, it is possible to apply higher selection intensities using the drought score system, and the selected lines can be further tested for grain yield in the wet season. (C) 2004 Elsevier B.V. All rights reserved.

Evaluation of eight spring onion genotypes, sulphur nutrition and soil-type effects with an electronic nose

Genotype, sulphur (S) nutrition and soil-type effects on spring onion quality were assessed using a 32-conducting polymer sensor E-nose. Relative changes in sensor resistance ratio (% dR/R) varied among eight spring onion genotypes. The % dR/R was reduced by S application in four of the eight genotypes. For the other four genotypes, S application gave no change in % dR/R in three, and increased % dR/R in the other. E-nose classification of headspace volatiles by a two-dimensional principal component analysis (PCA) plot for spring onion genotypes differed for S fertilisation vs. no S fertilisation. Headspace volatiles data set clusters for cv. 'White Lisbon' grown on clay or on sandy loam overlapped when 2.9 [Mahalanobis distance value (D2) = 1.6], or 5.8-(D2 = 0.3) kg S ha-1 was added. In contrast, clear separation (D2 = 7.5) was recorded for headspace volatile clusters for 0 kg S hd-1 on clay vs. sandy loam. Addition of 5.8 kg S ha-1 increased pyruvic acid content (mmole g-1 fresh weight) by 1.7-fold on average across the eight genotypes. However, increased S from 2.9 to 5.8 kg ha-1 did not significantly (P > 0.05) influence % dR/R, % dry matter (DM) or total soluble solids (TSS) contents, but significantly (P < 0.05) increased pyruvic acid content. TSS was significantly (P < 0.05) reduced by S addition, while % DM was unaffected. In conclusion, the 32-conducting polymer E-nose discerned differences in spring onion quality that were attributable to genotype and to variations in growing conditions as shown by the significant (P < 0.05) interaction effects for % dR/R.

Use of drought response index for identification of drought tolerant genotypes in rainfed lowland rice

Drought is a major constraint for rice production in the rainfed lowlands in Southeast Asia and Eastern India. The breeding programs for tainted lowland rice in these regions focus on adaptation to a range of drought conditions. However, a method of selection of drought tolerant genotypes has not been established and is considered to be one of the constraints faced by rice breeders. Drought response index (DRI) is based on grain yield adjusted for variation in potential yield and flowering date, and has been used recently, but its consistency among drought environments and hence its usefulness is not certain. In order to establish a selection method and subsequently to identify donor parents for drought resistance breeding, a series of experiments with 15 contrasting genotypes was conducted under well-watered and managed drought conditions at two sites for 5 years in Cambodia. Water level in the field was recorded and used to estimate the relative water level (WLREL) around flowering as an index of the severity of water deficit at the time of flowering for each entry. This was used to determine if DRI or yield reduction was due to drought tolerance or related to the amount of available water at flowering, i.e. drought escape. Grain yield reduction due to drought ranged from 12 to 46%. The drought occurred mainly during the reproductive phase, while four experiments had water stress from the early vegetative stage. There was significant variation for water availability around flowering among the nine experiments and this was associated with variation in mean yield reduction. Genotypic variation in DRI was consistent among most experiments, and genotypic mean DRI ranged from -0.54 to 0.47 (LSD 5% = 0.47). Genotypic variation in DRI was not related to WLREL around flowering in the nine environments. It is concluded that selection for DRI under drought conditions would allow breeders to identify donor lines with high drought tolerance as an important component of breeding better adapted varieties for the rainfed lowlands; two genotypes were identified with high DRI and low yield reduction and were subsequently used in the breeding program in Cambodia. (c) 2006 Elsevier B.V. All rights reserved.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Role of protein kinase C and NF-kappa B in proteolysis-inducing factor-induced proteasome expression in C2C12 myotubes

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4alpha-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 muM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 muM). Proteolysis-inducing factor-induced activation of PKCalpha, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCalpha (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCalpha (pKS1), which showed no activation of PKCalpha in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-kappaB (NF-kappaB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-kappaBalpha and an increase in nuclear binding of NF-kappaB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-kappaBalpha, induced by PIF, necessary for the release of NF-kappaB from its inactive cytosolic complex.

Targeting the glycoprotein 130 receptor subunit to control pain and inflammation

The glycoprotein 130 (gp130) is a shared signal-transducing-membrane-associated receptor for several hematopoietic cytokines. Its activation is implicated in pain and in a variety of diseases via signaling of proinflammatory cytokines. These include interleukin-6 (IL-6) subfamily cytokines, many of which play important roles in the pathogenesis of diseases such as rheumatoid arthritis, Castleman's disease, and Kaposi's sarcoma. Several strategies have been developed to block gp130-receptor-mediated signaling. These include the application of monoclonal antibodies, the creation of mutant form(s) of the gp130 with increased binding affinity for such ligands as IL-6/sIL-6R complex, and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor binding site(s). Other strategies include targeting gp130-mediated signaling pathways such as that involving signal transducer and activator of transcription-3. This review provides a summary of the latest research pertaining to the role of gp130 in the pathogenesis of inflammatory and other diseases in which the gp130 receptor is implicated. An overview of antagonists targeting the gp130 receptor is included with particular emphasis on their mechanism of action and their limitations and potential for therapeutic application.