Membrane and walls: who is master, who is servant?

Specialised plant cell types often locally modify their cell walls as part of a developmental program, as do cells that are challenged by particular environmental conditions. Modifications can include deposition of secondary cellulose, callose, cutin, suberin or lignin. Although the biosyntheses of cell wall components are more and more understood, little is known about the mechanisms that control localised deposition of wall materials. During metaxylem vessel differentiation, site-specific cell wall deposition is locally prevented by the microtubule depolymerising protein MIDD1, which disassembles the cytoskeleton and precludes the cellulose synthase complex from depositing cellulose. As a result, metaxylem vessel secondary cell wall appears pitted. How MIDD1 is tethered at the plasma membrane and how other cell wall polymers are locally deposited remain elusive. Casparian strips in the root endodermis represent a further example of local cell wall deposition. The recent discovery of the Casparian Strip membrane domain Proteins (CASPs), which are located at the plasma membrane and are important for the site-specific deposition of lignin during Casparian strip development, establishes the root endodermis as an attractive model system to study the mechanisms of localised cell wall modifications. How secondary modifications are modulated and monitored during development or in response to environmental changes is another question that still misses a complete picture.

Extreme heterochiasmy and nascent sex chromosomes in European tree frogs.

We investigated sex-specific recombination rates in Hyla arborea, a species with nascent sex chromosomes and male heterogamety. Twenty microsatellites were clustered into six linkage groups, all showing suppressed or very low recombination in males. Seven markers were sex linked, none of them showing any sign of recombination in males (r=0.00 versus 0.43 on average in females). This opposes classical models of sex chromosome evolution, which envision an initially small differential segment that progressively expands as structural changes accumulate on the Y chromosome. For autosomes, maps were more than 14 times longer in females than in males, which seems the highest ratio documented so far in vertebrates. These results support the pleiotropic model of Haldane and Huxley, according to which recombination is reduced in the heterogametic sex by general modifiers that affect recombination on the whole genome.

Interactions of Adeno-associated virus Rep78 protein with the host cell

Abstract : Adeno-associated virus (AAV) is a small DNA virus belonging to the familiy of Parvoviridae. Its genome contains two genes : the rep gene encoding four non structural proteins (Rep78, 68, 52 and 40) implicated in transcription, replication and site-specific integration of the viral DNA and the cap gene encoding three capsid proteins. AAV does not cause any disease, but is studied in view of its potential use to treat several diseases. An interesting property of AAV is its antiproliferative effect. Two elements of AAV can inhibit cell growth. Firstly, the single stranded viral DNA is recognized in cells as damaged DNA leading to either a G2 block or cell death depending on p53 status. Secondly, the two larger Rep proteins (Rep78 and 68) also arrest the cell cycle when they are expressed at high levels. Rep78 in particular induces a complete cell cycle arrest in all the phases, including S phase. Such a strong S phase arrest is rarely seen in other conditions. It was thus interesting to determine how Rep78 could induce it. We found that this strong block is the consequence of Rep78's effects on at least two pathways. Rep78 induces a DNA damage response by producing nicks in the cellular chromatin. Furthermore, Rep78 can bind to the cellular phosphatase Cdc25A and prevent its binding to its substrates CDK2 and CDK1, thus inhibiting its activity. A mutational analysis of Rep78 protein determined that its endonuclease activity is responsible for the DNA damage response and its zinc finger domain for Cdc25A inhibition. The combined expression of two mutants each defective for one of these activities, or these two activities obtained independently of Rep78, could restore the complete cell cycle block, indicating that these two effects of Rep78 are likely to explain completely the cell cycle block it induces. Secondly, the lack of pathogenicity of AAV, its broad range of infection and its ability to integrate site-specifically in human chromosome 19 make it an interesting potential vector for gene therapy. However site-specific integration is only possible in the presence of Rep78/68 whose gene is removed in recombinant AAV vectors. In this part of the study, we tried to introduce Rep protein separately from recombinant AAV vectors to promote their site-specific integration. For that purpose, a fusion protein, TAT-Rep, comprising Rep78/68 joined to the human immunodeficiency virus Tat protein was produced. It had the ability to enter cells and remain active there for a short period. Its activity was sufficient to mediate transcription from the p5 promoter, second-strand synthesis of a recombinant AAV and probably site-specific integration. Résumé : Le virus associé à l'adénovirus (AAV) est un petit virus à ADN qui fait partie de la famille des Parvoviridae. Son génome contient deux gènes : le gène rep code pour quatre protéines (Rep78, 68, 52 et 40) qui participent à la transcription, la réplication et l'intégration du virus et le gène cap code pour les trois protéines de capside. AAV ne produit pas de maladie, mais pourrait au contraire être utilisé pour en soigner. Sa bénignité, sa capacité à infecter différents types de cellules et son intégration spécifique en font un vecteur potentiel pour la thérapie génique. Pour qu'il puisse s'intégrer spécifiquement, il a besoin de la protéine Rep78 ou 68, mais ce gène doit être enlevé des vecteurs pour la thérapie génique. Le but de la première partie de cette étude était d'introduire Rep78 ou 68 dans des cellules en même temps qu'un AAV recombinant, mais indépendamment afin de permettre une intégration spécifique. La stratégie utilisée était de produire une protéine de fusion (TAT-Rep) qui peut entrer dans des cellules si elle est présente dans leur milieu. Cette protéine entrait bien dans les cellules et y était active favorisant ainsi l'intégration spécifique. Une deuxième propriété d'AAV, son effet anti-prolifératif, est intéressante dans le cadre de certaines maladies comme le cancer. Deux éléments d'AAV en sont responsables. D'abord, son ADN simple brin active une réponse cellulaire à l'ADN endommagé et arrête les cellules en G2 ou provoque leur mort. De plus, la protéine Rep78 d'AAV peut fortement bloquer le cycle cellulaire à toutes les phases, même en phase S, ce qui est rare. C'est pourquoi nous avons essayé de comprendre cet effet. Nous avons remarqué que Rep78 doit agir sur deux fronts pour obtenir ce fort bloc. D'un côté, Rep78 introduit des coupures simple brin sur l'ADN de la cellule ce qui active une réponse cellulaire à l'ADN endommagé qui passe par ATM. D'un autre côté, Rep78 lie une phosphatase cellulaire, Cdc25A, et l'empêche ainsi de lier ses substrats CDK2 et CDK1 et donc d'être active. Finalement, à l'aide de mutants de Rep78, nous avons déterminé que l'activité endonuclease de Rep78 était nécessaire pour induire une réponse cellulaire via ATM et que le domaine C-terminal appelé «zinc finger » était responsable de la liaison avec Cdc25A. En co-exprimant deux mutants, qui n'ont chacun qu'un des effets de Rep78, ou en obtenant les deux effets de Rep78 indépendamment d'elle, nous avons obtenu un bloc complet du cycle cellulaire similaire à celui obtenu avec Rep78. Il est donc probable que ces deux effets de Rep78 sont suffisants pour expliquer comment elle arrive à arrêter le cycle cellulaire si efficacement.

Total exposure and exposure rate effects for alcohol and smoking and risk of head and neck cancer: a pooled analysis of case-control studies.

Although cigarette smoking and alcohol consumption increase risk for head and neck cancers, there have been few attempts to model risks quantitatively and to formally evaluate cancer site-specific risks. The authors pooled data from 15 case-control studies and modeled the excess odds ratio (EOR) to assess risk by total exposure (pack-years and drink-years) and its modification by exposure rate (cigarettes/day and drinks/day). The smoking analysis included 1,761 laryngeal, 2,453 pharyngeal, and 1,990 oral cavity cancers, and the alcohol analysis included 2,551 laryngeal, 3,693 pharyngeal, and 3,116 oval cavity cancers, with over 8,000 controls. Above 15 cigarettes/day, the EOR/pack-year decreased with increasing cigarettes/day, suggesting that greater cigarettes/day for a shorter duration was less deleterious than fewer cigarettes/day for a longer duration. Estimates of EOR/pack-year were homogeneous across sites, while the effects of cigarettes/day varied, indicating that the greater laryngeal cancer risk derived from differential cigarettes/day effects and not pack-years. EOR/drink-year estimates increased through 10 drinks/day, suggesting that greater drinks/day for a shorter duration was more deleterious than fewer drinks/day for a longer duration. Above 10 drinks/day, data were limited. EOR/drink-year estimates varied by site, while drinks/day effects were homogeneous, indicating that the greater pharyngeal/oral cavity cancer risk with alcohol consumption derived from the differential effects of drink-years and not drinks/day.

The application of NMR and MS methods for detection of adulteration of wine, fruit juices, and olive oil. A review

This review covers two important techniques, high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), used to characterize food products and detect possible adulteration of wine, fruit juices, and olive oil, all important products of the Mediterranean Basin. Emphasis is placed on the complementary use of SNIF-NMR (site-specific natural isotopic fractionation nuclear magnetic resonance) and IRMS (isotope-ratio mass spectrometry) in association with chemometric methods for detecting the adulteration.

Transgenic mice and their impact on kidney research.

The kidney is a key organ in the maintenance of ion and fluid homeostasis and specific transport systems localized along the nephron guarantee this function. Due to its large functional heterogeneity, experiments on the whole organ level cannot be easily performed, and thus more refined tools are needed, like for example the development of specific recombination systems to gain knowledge on the physiological role of single proteins implicated in ion transport. This review introduces the transgenic technology developed over the past decades, and then focuses on recent strategies for generating kidney-specific gene targeting, over-expression, and gene ablation in mice, that will help to understand the physiological role of proteins implicated in salt and water balance in the kidney.

Reconsolidation of the soil surface after tillage discontinuity, with and without cultivation, related to erosion and its prediction with RUSLE

Site-specific regression coefficient values are essential for erosion prediction with empirical models. With the objective to investigate the surface-soilconsolidation factor, Cf, linked to the RUSLE's prior-land-use subfactor, PLU, an erosion experiment using simulated rainfall on a 0.075 m m-1 slope, sandy loam Paleudult soil, was conducted at the Agriculture Experimental Station of the Federal University of Rio Grande do Sul (EEA/UFRGS), in Eldorado do Sul, State of Rio Grande do Sul, Brazil. Firstly, a row-cropped area was excluded from cultivation (March 1995), the existing crop residue removed from the field, and the soil kept clean-tilled the rest of the year (to get a degraded soil condition for the intended purpose of this research). The soil was then conventional-tilled for the last time (except for a standard plot which was kept continuously cleantilled for comparison purposes), in January 1996, and the following treatments were established and evaluated for soil reconsolidation and soil erosion until May 1998, on duplicated 3.5 x 11.0 m erosion plots: (a) fresh-tilled soil, continuously in clean-tilled fallow (unit plot); (b) reconsolidating soil without cultivation; and (c) reconsolidating soil with cultivation (a crop sequence of three corn- and two black oats cycles, continuously in no-till, removing the crop residues after each harvest for rainfall application and redistributing them on the site after that). Simulated rainfall was applied with a Swanson's type, rotating-boom rainfall simulator, at 63.5 mm h-1 intensity and 90 min duration, six times during the two-and-half years of experimental period (at the beginning of the study and after each crop harvest, with the soil in the unit plot being retilled before each rainfall test). The soil-surface-consolidation factor, Cf, was calculated by dividing soil loss values from the reconsolidating soil treatments by the average value from the fresh-tilled soil treatment (unit plot). Non-linear regression was used to fit the Cf = e b.t model through the calculated Cf-data, where t is time in days since last tillage. Values for b were -0.0020 for the reconsolidating soil without cultivation and -0.0031 for the one with cultivation, yielding Cf-values equal to 0.16 and 0.06, respectively, after two-and-half years of tillage discontinuation, compared to 1.0 for fresh-tilled soil. These estimated Cf-values correspond, respectively, to soil loss reductions of 84 and 94 %, in relation to soil loss from the fresh-tilled soil, showing that the soil surface reconsolidated intenser with cultivation than without it. Two distinct treatmentinherent soil surface conditions probably influenced the rapid decay-rate of Cf values in this study, but, as a matter of a fact, they were part of the real environmental field conditions. Cf-factor curves presented in this paper are therefore useful for predicting erosion with RUSLE, but their application is restricted to situations where both soil type and particular soil surface condition are similar to the ones investigate in this study.

Evidence against close linkage of the loci for fraXq of Martin-Bell syndrome and for factor IX.

Linkage between the loci for fraXq of Martin-Bell syndrome and factor IX was studied in nine families exhibiting this syndrome by means of a restriction fragment length polymorphism at the factor IX locus. Computer analysis of the data indicates there to be no evidence for close linkage between the syndrome and the factor IX locus.

Numerical simulation of ambient seismic wavefield modification caused by pore-fluid effects in an oil reservoir

We have modeled numerically the seismic response of a poroelastic inclusion with properties applicable to an oil reservoir that interacts with an ambient wavefield. The model includes wave-induced fluid flow caused by pressure differences between mesoscopic-scale (i.e., in the order of centimeters to meters) heterogeneities. We used a viscoelastic approximation on the macroscopic scale to implement the attenuation and dispersion resulting from this mesoscopic-scale theory in numerical simulations of wave propagation on the kilometer scale. This upscaling method includes finite-element modeling of wave-induced fluid flow to determine effective seismic properties of the poroelastic media, such as attenuation of P- and S-waves. The fitted, equivalent, viscoelastic behavior is implemented in finite-difference wave propagation simulations. With this two-stage process, we model numerically the quasi-poroelastic wave-propagation on the kilometer scale and study the impact of fluid properties and fluid saturation on the modeled seismic amplitudes. In particular, we addressed the question of whether poroelastic effects within an oil reservoir may be a plausible explanation for low-frequency ambient wavefield modifications observed at oil fields in recent years. Our results indicate that ambient wavefield modification is expected to occur for oil reservoirs exhibiting high attenuation. Whether or not such modifications can be detected in surface recordings, however, will depend on acquisition design and noise mitigation processing as well as site-specific conditions, such as the geologic complexity of the subsurface, the nature of the ambient wavefield, and the amount of surface noise.

Population-based registration of phenotypic anatomic and familial data for melanoma: results from a Swiss multicentric study

CONTEXT AND OBJECTIVES: A multicentric study was set up to assess the feasibility for Swiss cancer registries of actively retrieving 3 additional variables of epidemiological and a etiological relevance for melanoma, and of potential use for the evaluation of prevention campaigns. MATERIAL AND METHODS: The skin type, family history of melanoma and precise anatomical site were retrieved for melanoma cases registered in 5 Swiss cantons (Neuchâtel, St-Gall and Appenzell, Vaud and Wallis) over 3 to 6 consecutive years (1995-2002). Data were obtained via a short questionnaire administered by the physicians - mostly dermatologists - who originally excised the lesions. As the detailed body site was routinely collected in Ticino, data from this Cancer Registry were included in the body site analysis. Relative melanoma density (RMD) was computed by the ratio of observed to expected numbers of melanomas allowing for body site surface areas, and further adjusted for site-specific melanocyte density. RESULTS: Of the 1,645 questionnaires sent, 1,420 (86.3%) were returned. The detailed cutaneous site and skin type were reliably obtained for 84.7% and 78.7% of questionnaires, and family history was known in 76% of instances. Prevalence of sun-sensitive subjects and patients with melanoma affected first-degree relatives, two target groups for early detection and surveillance campaigns were 54.1% and 3.4%, respectively. After translation into the 4th digit of the International Classification of Diseases for Oncology, the anatomical site codes from printed (original information) and pictorial support (body chart from the questionnaire) concurred for 94.6% of lesions. Discrepancies occurred mostly for lesions on the upper, outer part of the shoulder for which the clinician's textual description was "shoulder blade". This differential misclassification suggests under-estimation by about 10% of melanomas of the upper limbs and an over-estimation of 5% for truncal melanomas. Sites of highest melanoma risk were the face, the shoulder and the upper arm for sexes, the back for men and the leg for women. Three major features of this series were: (1) an unexpectedly high RMD for the face in women (6.2 vs 4.2 in men), (2) the absence of a male predominance for melanomas on the ears, and (3) for the upper limbs, a steady gradient of increasing melanoma density with increasing proximity to the trunk, regardless of sex. DISCUSSION AND CONCLUSION: The feasibility of retrieving the skin type, the precise anatomical location and family history of melanoma in a reliable manner was demonstrated thanks to the collaboration of Swiss dermatologists. Use of a schematic body drawing improves the quality of the anatomical site data and facilitate the reporting task of doctors. Age and sex patterns of RMD paralleled general indicators of sun exposure and behaviour, except for the hand (RMD=0.2). These Swiss results support some site or sun exposure specificity in the aetiology of melanoma.

Effect of fertilizer-N application and seed coating with rhizobial inoculants on soybean yield in eastern Paraguay

Nitrogen removal in soybean grains at harvest may exceed biological N2 fixation, particularly if grain yields are as high as typically achieved on "Terra Rossa" soils of Eastern Paraguay. Applying N fertilizer or coating seeds with rhizobial inoculants that enhance nodulation may represent a way of balancing the N budget. However, the effects of such treatments appear to be highly site-specific. The objective of this study was to examine the effects of N application (N) and rhizobial inoculation (I) on nodulation, N accumulation and soybean yields in Eastern Paraguay. Field experiments were conducted in two consecutive soybean seasons. Dry conditions in the first year delayed sowing and reduced plant number m-2 and pod number plant-1. Grain yields were generally below 2 t ha-1 but the +N+I treatment increased yields by about 75%. In the second year favorable conditions resulted in yields of around 4 t ha-1 and the treatments had no effect. Nitrogen accumulation was higher in the first year and could therefore not explain the observed yield differences between years and treatment combinations. The positive effect of the +N+I treatment in year one was associated with a more rapid root growth which could have reduced susceptibility to intermittent drought stress. Nodule biomass decreased between flowering and pod setting stages in the +I treatment whereas further increases in nodule biomass in the -I treatment may have led to competition for assimilates between nodules and developing pods. Based on these preliminary results we conclude that N application and seed inoculation can offer short-term benefits in unfavorable years without negative effects on yield in favorable years.

The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach.

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

Optical crop sensor for variable-rate nitrogen fertilization in corn: i - plant nutrition and dry matter production

Variable-rate nitrogen fertilization (VRF) based on optical spectrometry sensors of crops is a technological innovation capable of improving the nutrient use efficiency (NUE) and mitigate environmental impacts. However, studies addressing fertilization based on crop sensors are still scarce in Brazilian agriculture. This study aims to evaluate the efficiency of an optical crop sensor to assess the nutritional status of corn and compare VRF with the standard strategy of traditional single-rate N fertilization (TSF) used by farmers. With this purpose, three experiments were conducted at different locations in Southern Brazil, in the growing seasons 2008/09 and 2010/11. The following crop properties were evaluated: above-ground dry matter production, nitrogen (N) content, N uptake, relative chlorophyll content (SPAD) reading, and a vegetation index measured by the optical sensor N-Sensor® ALS. The plants were evaluated in the stages V4, V6, V8, V10, V12 and at corn flowering. The experiments had a completely randomized design at three different sites that were analyzed separately. The vegetation index was directly related to above-ground dry matter production (R² = 0.91; p<0.0001), total N uptake (R² = 0.87; p<0.0001) and SPAD reading (R² = 0.63; p<0.0001) and inversely related to plant N content (R² = 0.53; p<0.0001). The efficiency of VRF for plant nutrition was influenced by the specific climatic conditions of each site. Therefore, the efficiency of the VRF strategy was similar to that of the standard farmer fertilizer strategy at sites 1 and 2. However, at site 3 where the climatic conditions were favorable for corn growth, the use of optical sensors to determine VRF resulted in a 12 % increase in N plant uptake in relation to the standard fertilization, indicating the potential of this technology to improve NUE.

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways.

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.