RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Diagnóstico biológico e molecular e análise da seqüência de nucleotídeos do gene da proteína capsidial de um isolado do Apple stem pitting virus

O Apple stem pitting virus (ASPV) foi detectado por RT-PCR em amostras de cultivares de pereiras européias (Pyrus communis L.) cvs. Starkrimson e Abate Fetel, e asiáticas (P. pyrifolia var. culta) cvs. Kousui e Housui coletadas no início do outono de 2003 em pomar da Estação Experimental da Embrapa Uva e Vinho, Vacaria, RS. Utilizando várias combinações de oligonucleotídeos, foram amplificados fragmentos de DNA de 269 e 1554 pb, este último contendo o gene completo (1131 nt) da proteína capsidial do ASPV. Outro fragmento amplificado de 291 pb compreende parte do gene da polimerase viral. Estes fragmentos constituem-se em um excelente instrumento de diagnóstico do ASPV em pereiras. A comparação das seqüências de nucleotídeos do gene da proteína capsidial do ASPV com seqüências do banco de dados GenBank, revelou identidades de 89% com seqüências de um isolado alemão de macieira e de 85 a 88% com isolados poloneses, de pereiras. A indicadora herbácea Nicotiana occidentalis cv. 37B, inoculada mecanicamente com extrato foliar da cv. Housui, apresentou lesões locais necróticas, necrose foliar marginal e das nervuras. O ASPV também foi detectado por dot-ELISA nas cvs. Abate Fetel e Kousui, na cv. Starkrimson por imunoblot, e em Pyronia veitchii (Trabut) Guill. por enxertia de borbulhas da cv. Abate Fetel infetada.

Determination of patulin in apple juice by HPLC using a simple and fast sample preparation method

The goal of this work was to develop a simple and rapid preparation method for patulin analysis in apple juice without previous clean-up. This method combined sonication and liquid extraction techniques and was used for determination of patulin in 37 commercial apple juices available on the market in the South of Brazil. The method performance characteristics were determined using a sample obtained in a local market fortified at five concentration levels of patulin and done in triplicates. The coefficient of variation for repeatability at the fortification level of 20.70µg.L-1 was 3.53 % and the recovery 94.63 %, respectively. The correlation coefficient was 0.9996 and agrees with the requirements for a linear analytical method value. The detection limit was 0.21µg.L-1 and the quantification limit 0.70 µg.L-1. Only three of the analyzed samples were upper the allowed level of 50.00 µg.L-1 recommended for the World Health Organization.

Apple, Nokia and RIM – Rivalry and Co-operation between Three Mobile Phone Corporations

The purpose of this paper is to gather enough evidence to speculate the future of Nokia, Rim and Apple. The thesis goes over the history, current events and business model of each company. This paper includes differences between the companies and co-operation and rivalry, such as patent infringement cases. The study is limited to smartphones and their future. The result of this study is that Apple will continue its steady increase in market share, while Nokia will first decrease and after the launch of the Windows Phone it will rise again. RIM‟s result has not been as good as in past years and it has lost market share. The decrease of share price may lead to acquisition by a company interested in RIM technology.

Spatial influence of physical and chemical parameters on management zone definition in apple orchards

ABSTRACT Precision agriculture adoption in Brazilian apple orchards is still incipient. This study aimed at evaluating the spatial variability of certain soil properties as soil density, soil penetration resistance, electrical conductivity, yield, and fruit quality in an apple orchard through digital mapping, as well as assessing the correlation between these factors by means of geostatistics, establishing management zones. Forty representative points were set within 2.5 hectares of apple orchard, wherein soil samples were collected and analyzed, besides measurements of fruit quality (Brix degree, size or diameter, pulp firmness and color) to generate an overall index quality. We concluded that the fruit quality indexes, when isolated, did not show strong spatial dependence, unlike the index of fruit quality (FQI), derived from a combination of these parameters, allowing orchard planning according to management zones based on quality.

Yritysten teknologisen keskittymisen tutkiminen patenttianalyysin avulla Case: Apple, HTC, Nokia, Samsung

Työssä tutkitaan neljän matkapuhelinvalmistajan teknologista keskittymistä patenttianalyysin avulla. Tavoitteena on tutkia, onko yritysten patentoinnissa eroja sekä mihin eri teknologian osa-alueisiin yritykset patentoivat ja mihin eivät. Tavoitteen taustalla on selvittää, voidaanko patenttiaineistosta saada viitteitä yrityksen ydinosaamisesta. Työ koostuu kolmesta rakenteellisesta osiosta. Teoria esittelee patentit ja patenttiviittaukset. Patenttiaineisto on kerätty Lappeenrannan teknillisen yliopiston patenttipalvelimelta ja sitä on analysoitu tietokanta- sekä tilastollisen käsittelyn ohjelmalla. Tuloksista tehdyt analyysit yhdistetään teoriaan, jonka pohjalta tehdään johtopäätöksiä sekä suoritetaan vertailua yritysten kesken liittyen tutkimuskysymyksiin. Lopuksi arvioidaan aineiston ja tulosten luotettavuutta sekä esitetään mahdollisia jatkotutkimusaiheita.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

Sexual dysfunction in obese women is more affected by psychological domains than that of non-obese

Abstract PURPOSE: To compare differences in the occurrence and changed domains of sexual dysfunction in obese and non-obese Brazilian women. METHODS: Female Sexual Function Index, based on six domains, to investigate 31 sexual dysfunction incidence for obese compared to 32 non-obese women, was used. Statistical analysis using ANOVA and MANOVA were performed to compare total scores of Female Sexual Function Index among groups and to identify the differences among domains, Student t -test was used. Statistical significant level was established for all tests for p<0.05. RESULTS: No difference in female sexual dysfunction frequency between obese (25.8%) and non-obese women (22.5%) was found. However, an important distinction in which aspects of sexual life were affected was found. While the obese group was impaired in three domains of sexual life (desire, orgasm, and arousal), in the control group five aspects were dysfunctional (desire, orgasm, arousal, pain and lubrication). Future research exploring psychological outcomes in obese females, such as body image and measures of positive and negative effect, might better characterize the female sexual dysfunction in this group. CONCLUSIONS: Obesity does not appear to be an independent factor for allow quality of female sexual life. However, disturbance associated to obesity indicates a low frequency of disorder in physical domains, suggesting that psychological factors seem to be mainly involved in the sexual dysfunction in obese women.

Floristic composition and growth of weeds under custard apple (Annona squamosa) progenies

Weeds have a negative influence on several fruit tree characteristics, such as yield, making it difficult to management practices in orchards. Alternative weed management methods, aiming to reduce the use of herbicides, have become attractive since herbicides are costly and cause environmental degradation. The use of cultivars with greater competitive ability against weeds has attracted international attention. The objective of this work was to evaluate the floristic composition and growth of weeds under the canopies of irrigated custard apple tree progenies. Twenty halfsibling progenies around three years of age were evaluated in a random block design with five replicates and four plants per plot. A circle with a 0.5 m² area was established around the trunk of each plant. Floristic composition, fresh matter, and dry matter mass of the above-ground part of the weeds, were evaluated in this area. Root collar and canopy diameters, as well as leaf area of the progenies were also evaluated. Fifty-eight weed species were recorded. The five weed families with the most species were Leguminosae, Convolvulaceae, Euphorbiaceae, Malvaceae and Sterculiaceae, in decreasing order. The number of weed species per plot ranged from 6 to 18, but there was no difference between the mean percentages of different weeds under the canopies of the progenies. The lowest weed fresh and dry matter masses occurred in progenies JG1 and SM8, respectively. There were no differences between progenies with regard to root collar diameter and leaf area; however, one of the lowest weed dry matter yields was observed under the canopy of progeny FE4, which showed the largest canopy diameter.

Connexin domains relevant to the chemical gating of gap junction channels

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with sparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL1 and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.