Social determinants of tuberculosis evaluation among household contacts: a secondary analysis.

Setting: Seven public sector tuberculosis (TB) units and surrounding communities in Kampala, Uganda. Objective: To evaluate the influence of household-level socio-economic characteristics on completion of TB evaluation during household contact investigation. Design: A cross-sectional study nested within the control arm of a randomized, controlled trial evaluating home-based sputum collection and short messaging service communications. We used generalized estimating equations to estimate the association between completion of TB evaluation and socio-economic determinants. Results: Of 116 household contacts referred to clinics for TB evaluation, 32 (28%) completed evaluation. Completing evaluation was strongly clustered by household. Controlling for individual symptoms, contacts from households earning below-median income (adjusted risk ratio [aRR] 0.28, 95%CI 0.09-0.88, P = 0.029) and contacts from households in which the head of household had no more than primary-level education (aRR 0.40, 95%CI 0.18-0.89, P = 0.025) were significantly less likely to complete evaluation for TB. Conclusion: Socio-economic factors such as low income and education increase the risk that household contacts of TB patients will experience barriers to completing TB evaluation themselves. Further research is needed to identify specific mechanisms by which these underlying social determinants modify the capability and motivation of contacts to complete contact investigation.

Contexte : Sept unités de tuberculose (TB) du secteur public et les communautés qui les entourent à Kampala, Ouganda.Objectif : Evaluer l'influence des caractéristiques socioéconomiques des foyers sur l'achèvement de la recherche de TB lors de l'investigation des contacts domiciliaires.Schéma : Une étude transversale au sein du bras témoin d'un essai randomisé, contrôlé, évaluant le recueil de crachats à domicile et les communications par messagerie SMS. Nous avons utilisé des équations d'estimations généralisées afin d'estimer l'association entre l'achèvement de l'évaluation de la TB et les déterminants socioéconomiques.Résultats : Parmi les 116 contacts domiciliaires qui ont été référés à des centres de santé pour un bilan de TB, 32 (28%) ont achevé cette évaluation. L'achèvement de l'évaluation a été fortement regroupé par foyer. En contrôlant les symptômes individuels, les contacts des foyers ayant un revenu inférieur à la médiane (ratio de risque ajusté [RRa] 0,28 ; IC95% 0,09­0,88 ; P = 0,029) et les contacts des foyers dont le niveau d'instruction du chef ne dépassait pas l'école primaire (RRa 0,40 ; IC95% 0,18­0,89 ; P = 0,025) ont été significativement moins susceptibles de terminer le bilan de TB.Conclusion : Les facteurs socioéconomiques tels qu'un faible revenu et un faible niveau d'études augmentent le risque que les contacts domiciliaires de patients TB soient confrontés à des obstacles à l'achèvement de l'évaluation de la TB. Davantage de recherche est nécessaire pour identifier les mécanismes spécifiques par lesquels des déterminants sociaux sous-jacents modifient la capacité et la motivation des contacts à terminer leur bilan.

Marco de Referencia: Siete unidades de tuberculosis (TB) del sector público y las comunidades aledañas en Kampala, Uganda.Objetivo: Evaluar la influencia de las características socioeconómicas de los hogares en la compleción de la evaluación de la TB durante la investigación de contactos domiciliarios.Método: Fue este un estudio transversal anidado en la rama de referencia de un ensayo clínico aleatorizado, que evaluaba la recogida de esputo en los hogares y las comunicaciones por servicio de SMS. Se aplicaron ecuaciones de estimación generalizadas con el fin de apreciar la asociación entre la compleción de la investigación de la TB y los determinantes socioeconómicos.Resultados: Se remitieron a los consultorios 116 contactos domiciliarios para investigación de la TB y 32 completaron la evaluación (28%). La compleción de la investigación exhibió una importante distribución en conglomerados por hogares. Al ajustar con respecto a los síntomas individuales, se observó que era mucho menos probable que se completase la investigación de la TB en los contactos de hogares con un ingreso familiar por debajo de la mediana (razón de riesgos ajustada [aRR] 0,28; IC95% 0,09­0,88; P = 0,029) y en los contactos de hogares cuya cabeza de familia no tenía un grado de instrucción superior al nivel primario (aRR 0,40; IC95% 0,18­0,89; P = 0,025).Conclusión: Los factores socioeconómicos como un bajo ingreso y un grado inferior de instrucción aumentan el riesgo de que los contactos domiciliarios encuentren obstáculos propios para completar la investigación de TB. Se precisan nuevas investigaciones que definan los mecanismos mediante los cuales estos determinantes sociales subyacentes modifican la capacidad y la motivación de las personas a finalizar la investigación de contactos.

'Something so hard': a mixed-methods study of home sputum collection for tuberculosis contact investigation in Uganda.

BACKGROUND: Home sputum collection could facilitate prompt evaluation and diagnosis of tuberculosis (TB) among contacts of patients with active TB. We analyzed barriers to home-based collection as part of an enhanced intervention for household TB contact investigation in Kampala, Uganda. DESIGN: We conducted a convergent mixed-methods study to describe the outcomes of home sputum collection in 91 contacts and examine their context through 19 nested contact interviews and two focus group discussions with lay health workers (LHWs). RESULTS: LHWs collected sputum from 35 (39%) contacts. Contacts reporting cough were more likely to provide sputum than those with other symptoms or risk factors (53% vs. 15%, RR 3.6, 95%CI 1.5-2.8, P < 0.001). Males were more likely than females to provide sputum (54% vs. 32%, RR 1.7, 95%CI 1.0-2.8, P = 0.05). Contacts said support from the index patient and the convenience of the home visit facilitated collection. Missing containers and difficulty producing sputum spontaneously impeded collection. Women identified stigma as a barrier. LHWs emphasized difficulty in procuring sputum and discomfort pressing contacts to produce sputum. CONCLUSIONS: Home sputum collection by LHWs entails different challenges from sputum collection in clinical settings. More research is needed to develop interventions to mitigate stigma and increase success of home-based collection.

Integrating home HIV counselling and testing into household TB contact investigation: a mixed-methods study.

Setting: Community health workers (CHWs) increasingly deliver community-based human immunodeficiency virus (HIV) counselling and testing (HCT) services. Less is known about how this strategy performs when integrated with household tuberculosis (TB) contact investigations. Objective: We conducted a prospective mixed-methods study to evaluate the feasibility and quality of CHW-facilitated, home-based HCT among household TB contacts. Design: CHWs visited households of consenting TB patients to screen household contacts for TB and HIV. They performed HIV testing using a serial enzyme-linked immunosorbent assay rapid-antibody testing algorithm. Laboratory technicians at health facilities re-tested the samples and coordinated quarterly HIV panel testing for CHWs. We conducted focus group discussions (FGDs) with CHWs on their experiences in carrying out home-based HCT. Results: Of 114 household contacts who consented to and underwent HIV testing by CHWs, 5 (4%) tested positive, 108 (95%) tested negative, and 1 (1%) had indeterminate results; 110 (96%) samples had adequate volume for re-testing. Overall agreement between CHWs and laboratory technicians was 99.1% (κ = 0.90, 95%CI 0.71-1.00, P < 0.0001). In FGDs, CHWs described context-specific social challenges to performing HCT in a household setting, but said that their confidence grew with experience. Conclusion: Home-based HCT by CHWs was feasible among household TB contacts and produced high-quality results. Strategies to address social challenges are required to optimize yield.

Contexte : Les travailleurs de santé communautaire (CHW) offrent de plus en plus de services de conseil et de test communautaires relatifs au virus de l'immunodéficience humaine (HCT). On sait moins quel est le degré de performance de cette stratégie quand elle est intégrée à des visites à domicile à la recherche de contacts de tuberculose (TB).Objectif : Nous avons réalisé une étude prospective à méthodes variées afin d'évaluer la faisabilité et la qualité de CHW à domicile, facilité par des CHW dans les foyers des contacts de TB.Schéma : Les CHW ont visité les foyers des patients TB consentants afin de dépister les contacts domiciliaires de TB et du virus de l'immunodéficience humaine (VIH). Ils ont réalisé des tests VIH grâce à un algorithme de test rapide de recherche d'anticorps en série par titrage avec immunoadsorbant lié à une enzyme. Les techniciens de laboratoire des structures de santé ont re-testé les échantillons et coordonné un test VIH en groupe trimestriel pour les CHW. Nous avons réalisé des discussions en groupe focal (FGD) avec les CHW à propos de leurs expériences de HCT à domicile.Résultats : Ont été consentants 114 contacts domiciliaires qui ont été testés à la recherche du VIH par les CHW : 5 (4%) ont eu un test positif, 108 (95%) ont eu un test négatif et 1 seul (1%) a eu des résultats indéterminés ; 110 (96%) échantillons avaient un volume suffisant pour un deuxième test. Au total, l'accord entre les CHW et les techniciens de laboratoire a été de 99,1% (κ = 0,90 ; IC95% 0,71­1,00 ; P < 0,0001). Lors des FGD, les CHW ont décrit les défis sociaux spécifiques du contexte de la réalisation du HCT dans le cadre d'un foyer, mais ont affirmé que leur confiance en eux avait augmenté avec leur expérience.Conclusion : Le HCT à domicile par les CHW s'est avéré faisable parmi les contacts domiciliaires de TB et a produit des résultats de très bonne qualité. Des stratégies visant à résoudre les défis sociaux sont requises afin d'optimiser le rendement.

Marco de referencia: Los agentes de salud comunitarios (CHW) prestan cada vez con mayor frecuencia servicios de asesoramiento y pruebas de detección del virus de la inmunodeficiencia humana (HCT) en las comunidades. Se conoce poco sobre la eficacia de esta estrategia cuando se integra en la investigación de contactos domiciliarios de los pacientes con tuberculosis (TB).Objetivo: Se llevó a cabo un estudio prospectivo con métodos mixtos, con el objeto de evaluar la factibilidad y la calidad de los servicios de HCT prestados por los CHW a los contactos de los casos de TB en los hogares.Método: Los CHW visitaron los hogares de los pacientes con TB que dieron su consentimiento, con el fin de realizar el HCT en los contactos domiciliarios. Los CHW practicaron la investigación de la infección por el virus de la inmunodeficiencia humana (VIH) mediante un algoritmo de pruebas rápidas seriadas de anticuerpos de tipo inmunoabsorbente ligado a la enzima. Los auxiliares de laboratorio en los establecimientos de salud practicaban de nuevo las pruebas en las muestras y coordinaban la realización trimestral de series de pruebas por parte de los CHW. Se realizaron sesiones de grupos de opinión (FGD) con estos profesionales, a fin de compartir sus experiencias en HCT en los hogares.Resultados: Tras recibir su consentimiento, los CHW practicaron las pruebas del VIH a 114 contactos domiciliarios. Cinco contactos obtuvieron un resultado positivo (4%), en 108 el resultado fue negativo (95%) y en un caso el resultado fue indeterminado (1%). El volumen de 110 muestras (96%) fue suficiente para repetir las pruebas. La concordancia global entre los CHW y los auxiliares de laboratorio fue 99,1% (κ = 0,90; IC95% 0,71­1,00; P < 0,0001). En las FGD, los CHW describieron las dificultades sociales específicas del contexto que tuvieron que afrontar al prestar estos servicios en los hogares, pero afirmaron que con la práctica habían adquirido mayor confianza.Conclusión: La práctica domiciliaria del HCT a los contactos de los casos de TB por parte de los CHW fue factible y se obtuvieron resultados de gran calidad. Se precisan estrategias que respondan a las dificultades sociales encontradas con el propósito de optimizar el rendimiento.

Patterns of usage and preferences of users for tuberculosis-related text messages and voice calls in Uganda.

BACKGROUND: Little information exists about mobile phone usage or preferences for tuberculosis (TB) related health communications in Uganda. METHODS: We surveyed household contacts of TB patients in urban Kampala, Uganda, and clinic patients in rural central Uganda. Questions addressed mobile phone access, usage, and preferences for TB-related communications. We collected qualitative data about messaging preferences. RESULTS: We enrolled 145 contacts and 203 clinic attendees. Most contacts (58%) and clinic attendees (75%) owned a mobile phone, while 42% of contacts and 10% of clinic attendees shared one; 94% of contacts and clinic attendees knew how to receive a short messaging service (SMS) message, but only 59% of contacts aged 45 years (vs. 96% of contacts aged <45 years, P = 0.0001) did so. All contacts and 99% of clinic attendees were willing and capable of receiving personal-health communications by SMS. Among contacts, 55% preferred detailed messages disclosing test results, while 45% preferred simple messages requesting a clinic visit to disclose results. CONCLUSIONS: Most urban household TB contacts and rural clinic attendees reported having access to a mobile phone and willingness to receive TB-related personal-health communications by voice call or SMS. However, frequent phone sharing and variable messaging abilities and preferences suggest a need to tailor the design and monitoring of mHealth interventions to target recipients.

Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance.

The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2 O2 ) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells.

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione.

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.

Confocal imaging of glutathione redox potential in living plant cells.

Reduction-oxidation-sensitive green fluorescent protein (roGFP1 and roGFP2) were expressed in different sub-cellular compartments of Arabidopsis and tobacco leaves to empirically determine their performance as ratiometric redox sensors for confocal imaging in planta. A lower redox-dependent change in fluorescence in combination with reduced excitation efficiency at 488 nm resulted in a significantly lower dynamic range of roGFP1 than for roGFP2. Nevertheless, when targeted to the cytosol and mitochondria of Arabidopsis leaves both roGFPs consistently indicated redox potentials of about -320 mV in the cytosol and -360 mV in the mitochondria after pH correction for the more alkaline matrix pH. Ratio measurements were consistent throughout the epidermal cell layer, but results might be attenuated deeper within the leaf tissue. Specific interaction of both roGFPs with glutaredoxin in vitro strongly suggests that in situ both variants preferentially act as sensors for the glutathione redox potential. roGFP2 targeted to plastids and peroxisomes in epidermal cells of tobacco leaves was slightly less reduced than in other plasmatic compartments, but still indicated a highly reduced glutathione pool. The only oxidizing compartment was the lumen of the endoplasmic reticulum, in which roGFP2 was almost completely oxidized. In all compartments tested, roGFP2 reversibly responded to perfusion with H(2)O(2) and DTT, further emphasizing that roGFP2 is a reliable probe for dynamic redox imaging in planta. Reliability of roGFP1 measurements might be obscured though in extended time courses as it was observed that intense irradiation of roGFP1 at 405 nm can lead to progressive photoisomerization and thus a redox-independent change of fluorescence excitation ratios.

[Bone formation in the ureter. Osseous metaplasia of an obstructed ureter]. / Der Harnleiterknochen. Knöcherne Metaplasie eines obstruierten Harnleiters.

Bone formation outside the skeleton is well known. Yet, ossification within the urogenital tract is rare. The case of a patient with distal ureteral obstruction, hydronephrosis, and subsequent circular osseous metaplasia of the ureter is presented. Open transperitoneal right-sided nephrectomy with partial resection of the ureter was performed. Intraoperatively and in histopathologic examination circular bone formation adjacent to the ureteral wall was detected. There was no evidence of malignancy. Early animal experiments in the twentieth century showed that bone metaplasia may be induced by epithelium of the urinary tract under certain conditions such as ischemia, inflammation, necrosis, sclerosis, and trauma. Osseous metaplasia may therefore be considered a differential diagnosis of calcification of the upper urinary tract in the absence of urolithiasis.

Phylogenetic analysis of bluetongue virus genome segment 6 (encoding VP5) from different serotypes.

Full-length cDNAs were sequenced for genome segment 6 from representative isolates of all 24 bluetongue virus (BTV) serotypes. Segment 6 is 1635 to 1645 nucleotides in length with a single open reading frame in all isolates. The deduced VP5 protein sequence is 526 amino acids long for most BTVs, except BTV-15 (isolate number RSArrrr/15), which is 527 amino acids long, and BTV-12 (isolate number RSArrrr/12) and BTV-22 (isolate number Nig1982/11), which were 529 amino acids long. Sequence comparisons have revealed the level of genetic diversity in segment 6, between different BTV serotypes and between isolates within a single serotype. The resulting sequences can be used to design RT-PCR primers for amplification and identification of segment 6 from new BTV isolates, providing potentially valuable diagnostic and research tools. These data are available on the international databases and accession numbers are listed on the website of the Institute for Animal Health (iah.bbsrc.ac.uk/dsRNA_virus_proteins/ btv_sequences.htm).

Completion of the sequence analysis and comparisons of genome segment 2 (encoding outer capsid protein VP2) from representative isolates of the 24 bluetongue virus serotypes.

Bluetongue (BT) is a non-contagious, arthropod-transmitted viral disease of domestic and wild ruminants. It is caused by bluetongue virus (BTV), a double-stranded (ds) RNA virus that is classified within the genus Orbivirus, family Reoviridae. There are at least twenty-four serotypes of BTV worldwide, five of which (1, 2, 4, 9 and 16) have been identified recently in Europe. BTV infects ruminants and its distribution throughout temperate and tropical regions of the world is dependent on the activity and abundance of certain vector-competent species of Culicoides midge. The outer capsid protein VP2 of BTV is a major protective antigen and the primary determinant of virus serotype. For the first time, the authors have completed the sequence analysis of full-length VP2 genes from the reference strains of each of the 24 BTV serotypes and their amino acid sequences were deduced. Multiple alignment of the VP2 gene (protein) sequences revealed that the level of nucleotide (amino acid) sequence variation between serotypes ranged from 29% (23%) to 59% (73%), confirming that segment 2/VP2 is the most variable BTV gene/protein. Phylogenetic analysis of VP2 grouped together the BTV types that are known to cross-react serologically. Low identity between types was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralisation. The sequence data represent the completion of an important step in the creation of a comprehensive BTV sequence database, which will support more rapid molecular methods for diagnosis and identification of BTV 'types', as well as continuing molecular epidemiology and surveillance studies of BTV.

Cell-specific measurement of cytosolic glutathione in poplar leaves.

The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by high-performance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 mm for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial gamma-glutamylsynthetase (gamma-ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.

Quantitative in vivo measurement of glutathione in Arabidopsis cells.

A new, non-destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH-bimane (GSB) conjugate. At low (10-100 microM) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labelled low molecular-weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis-Menten kinetics with an apparent KM of 40 microM and Vmax of 470 micromol lcyt -1 min-1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of gamma-glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830-942 nmol g-1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 +/- 3% cytoplasm was determined by manual segmentation, and a value of 37 +/- 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 +/- 0.3 and 3.2 +/- 0.3 mM for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 +/- 0.5 and 3.5 +/- 0.7 mM, respectively.

Confocal imaging of metabolism in vivo: pitfalls and possibilities.

Confocal laser scanning microscopy (CLSM) has had wide application in morphological studies and ion imaging in plants, but little impact so far on biochemical investigations. This position is likely to change as the range of fluorescent probes increases. To illustrate the type of kinetic information that can be obtained using CLSM in an intact, living system, an analysis has been made of the two-step detoxification of monochlorobimane (MCB) following conjugation to glutathione (GSH) by a glutathione S-transferase in the cytoplasm and vacuolar sequestration of the fluorescent glutathione S-bimane (GSB) by a glutathione S-conjugate (GSX) pump. Fluorescence from the cytoplasm and vacuole of individual trichoblasts and atrichoblasts was measured from time-series of (x, y) optical sections in the elongation zone of Arabidopsis root tips. Intensity changes were calibrated and converted to amounts using compartment volumes, measured by stereological techniques. The data were well described using pseudo-first-order kinetics for the conjugation reaction and either Michaelis-Menten kinetics (Model I), or, as the GSX-pump was operating close to V(max), a pseudo-zero-order reaction (Model II), for the GSX-pump. Analysis of 15 individual cells from two roots gave [GSH](cyt) in the range 1.8-4 mM. GST activity was relatively constant on a cell basis in one root, but increased markedly in the other, giving a net increase in conjugation activity as cells progressed through the elongation zone. In contrast, GSX-pump activity increased in parallel with the increase in cell size in both roots, effectively maintaining a constant transport activity per unit root length or estimated vacuole surface area.

Glutathione biosynthesis in Arabidopsis trichome cells.

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode gamma-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238+/-25, 80+/-2, and 144+/-19 microM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH](cyt) is reduced to 47+/-5 microM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Novel mutations of the von hippel-lindau tumor-suppressor gene and rare DNA hypermethylation in renal-cell carcinoma cell lines of the clear-cell type.

Renal-cell carcinoma (RCC) is the most common neoplasm of the kidney, accounting for about 3% of all adult malignancies. Histopathologically, 80% of all cases can be classified as clear-cell RCC. Of these, approximately 55% to 70% are associated with mutations in the von Hippel-Lindau (VHL) tumor-suppressor gene. Here, new mutations of the VHL gene were defined by the use of temperature gradient gel electrophoresis and subsequent sequencing. In addition, DNA hypermethylation, an alternative mechanism of VHL gene silencing, was evaluated by methylation-specific PCR. Twenty-six clear-cell, 3 chromophilic, and 2 chromophobic RCC cell lines were analyzed. Among the clear-cell RCC cell lines tested, 12 (47%) contained 13 mutations overall: 8 (62%) in exon 1, 3 (23%) in exon 2, and 2 (15%) in exon 3. Ten of these mutations have thus far not been described. All single base pair changes were transversions. Six mutations led to alteration of a single amino acid. Seven mutations generated a frameshift or a stop codon. One cell line contained a complex duplication of 36 bp. All cell lines with mutations showed loss of heterozygosity in the VHL gene. No mutations could be detected in the chromophilic or chromophobic RCC samples. Significant hypermethylation was not observed in any of the cell lines. These data provide further evidence that distinct mutations in the VHL gene are a characteristic feature of clear-cell RCC. In contrast, hypermethylation of the gene is probably a rare event. The high frequency of transversion mutations suggests a role for exogenous carcinogens in the etiology of clear-cell RCCs.

Measurement of glutathione levels in intact roots of Arabidopsis.

Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S-bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15-20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S-conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2-3 mM in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell-cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.

Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two-photon laser scanning microscopy.

Two-photon laser scanning microscopy (TPLSM) was used to directly measure glutathione (GSH) as its fluorescent glutathione S-bimane conjugate (GSB) in developing root hair cells (trichoblasts) and non-root hair cells (atrichoblasts) of intact Arabidopsis roots. In comparison to confocal microscopy, TPLSM showed more detail deep within the tissue with less signal attenuation. The total level of GSB labelling reached a plateau after 60 min in both trichoblasts and atrichoblasts, reflecting depletion of GSH. GSB was formed initially in the cytoplasm and was subsequently transported into the vacuole. The volume ratio of vacuole to cytoplasm was determined using the Cavalieri estimator of volume and used to calculate the amount of GSB per volume of cytoplasm in each cell type. At the end of the time-course the cytoplasmic concentration of GSB was 2.7 +/- 0.5 mM (n = 5) in trichoblasts and 5.5 +/- 0.8 mM (n = 5) in atrichoblasts. In trichoblasts this value represents the initial concentration of GSH in the cytoplasm. Labelling of roots with monochlorobimane (MCB) on ice led to the formation of GSB in the cytoplasm, but prevented vacuolar sequestration. After washing prelabelled roots and transfer to room temperature, vacuolar transport resumed. Although no free MCB was present the total amount of GSB in atrichoblasts increased further, indicating that the higher values recorded in the atrichoblasts might reflect additional symplastic transport and sequestration of GSB from neighbouring cells.

The influence of fibre in the diet on growth rates and the digestibility of nutrients in the greater cane rat (Thryonomys swinderianus).

The greater cane rat Thryonomys swinderianus is a coprophagous rodent in which fermentation occurs in the large caecum. The extent to which a 45% increase in the fibre component of the diet influenced growth rates of cane rats and the digestibility of nutrients and energy was investigated in two feeding trials. Higher fibre levels in the diet reduced the digestibility of dry matter, protein and fat, while animals digested fibre components (neutral-detergent fibre, acid detergent fibre, hemicellulose and cellulose) with a comparable efficiency to those maintained on a low fibre diet. In one of the trials animals fed the high fibre diet exhibited significantly lower growth rates than animals fed the low fibre diet. Digestibility coefficients of the cane rats for neutral-detergent fibre and protein seem to be intermediate to high when compared to reported values for the porcupine, guinea-pig, degu and rabbit. It is suggested that the ability of cane rats to utilise large quantities of fibre enable them to survive periods when only dry grass is available.

Development of a mouse model system, coding assignments and identification of the genome segments controlling virulence of African horse sickness virus serotypes 3 and 8.

Attenuated (att) and wild type (wt) strains of the nine AHSV serotypes were evaluated for virulence in adult Balb C mice. Although most were avirulent in this system, isolates of AHSV 1att, 3wt, 3att, 4wt, 5att, 7att and 8att caused some mortality when administered via an intranasal route. After plaque cloning, only the attenuated vaccine strain of AHSV 7att caused any mortality via an intravenous route. AHSV 3att and AHSV 8wt were virulent (V) and avirulent (AV) (respectively) in the mouse model and were selected as parental strains for production of genome segment reassortants. These progeny virus strains were plaque cloned, then characterised to identify the genome segments that influence virulence of AHSV in the mouse model. Three virulence phenotypes were observed: fully virulent (V); fully avirulent (A); and a novel intermediate virulence (N) not expressed by either parental strain. Genome segment 2 (encoding outer capsid protein VP2) from the avirulent parent appeared to have a controlling influence in production of the A phenotype. Reassortants with the V phenotype all contained segment 2 from the virulent parent, however in each case they also contained genome segments 5 and 10, also from AHSV 3 (V). Genome segments 5 and 10 encode the smaller outer capsid protein VP5 and the non structural proteins NS3/NS3a, respectively. A combination of genome segments 2, 5 and 6 from the avirulent parent and segment 10 from the virulent parent were found in each of the virus strains with the N phenotype. However, comparison of two reassortants (A79 and A790), which differ only in a single segment, showed that replacement of genome segment 10 from the avirulent parent with that from the virulent parent, conferred the N phenotype on A790.