The Arabidopsis THADA homologue modulates TOR activity and cold acclimation.

Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor-kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants. We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation. Depletion of the AtTHADA gene caused complete or partial loss of full-length mRNA, respectively, and significant retardation of growth under non-stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low-temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de-regulation of TOR activity as one determinant for sensitivity towards low-temperatures. Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR.

Implementing carrier screening for cystic fibrosis outside the clinic: ethical analysis in the light of the personalist view.

BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive genetic disease. Two models for screening CF are normally used: newborn screening and population-based CF carrier screening. In turn, there are three main models of population-based CF carrier screening: prenatal carrier screening, preconception carrier screening, and carrier screening outside clinical settings. AIM: To evaluate, in the light of the personalist view, the use of carrier screenings for CF outside the clinic, i.e. in non-clinical settings, such as school and workplaces. METHODS: Analysis has been carried out according to the "Personalist approach" (also called "Triangular model"), an ethical method for performing ethical analysis within HTA process. It includes factual, anthropological and ethical data in a ''triangular'' normative reflection process. FINDINGS: Implementing carrier screening for cystic fibrosis outside the clinical settings allows acquisition of knowledge for informing reproductive choices, that can be considered as valuable; benefit-risk ratio seems to be not much favorable; autonomous and responsible decisions can be taken only under certain conditions; economic advantage is difficult to determine; therefore, from a personalist view, implementing carrier screenings outside the clinic seems not to be ethically justified. CONCLUSIONS: In accordance with the personalist perspective, public health programs providing carrier screenings outside the clinic should not be implemented.

[Genetic test for cancer and intra-family communication: freedom vs. responsibility]. / Test genetici in oncologia e comunicazione intrafamiliare: autonomia vs responsabilità.

Genetic tests affect not only single patients but also their genetic relatives. In some cases, they in fact allow to acquire information not only about a single patient, but also about those who are genetically linked (genetic relatives). By appealing to the principle of autonomy, the patient can refuse to be informed of the test result, or to inform their relatives on the risk of a pathology. How might the relatives' right to know be reconciled with the will of a patient who refuses to know or to inform? Among the large number of moral dilemmas that this field can raise, the article aims to reply to the above mentioned question and to analyse in depth some aspects of intra-family communication within the field of genetic tests for cancer.

Ethical evaluation of risks related to living donor transplantation programs.

The shortage of available cadaveric organs for transplantation and the growing demand has incresed live donation. To increase the number of transplantations from living donors, programs have been implemented to coordinate donations in direct or indirect form (cross-over, paired, and domino chain). Living donors with complex medical conditions are accepted by several transplantation programs. In this way, the number of transplants from living has exceeded that from cadaver donors in several European countries. No mortality has been reported in the case of lung, pancreas, or intestinal Living donations, but the perioperative complications range from 15% to 30% for pancreas and lung donors. In living kidney donors, the perioperative mortality is 3 per 10,000. Their frequency of end-stage renal disease does not exceed the United States rate for the general population. However, long-term follow-up studies of living donors for kidney transplantations have several limitations. The frequency of complications in live donor liver transplantation is 40%, of these, 48% are possibly life-threatening according to the Clavien classification. Residual disability, liver failure, or death has occurred in 1% of cases. The changes in live donor acceptance criteria raise ethical issues, in particular, the physician's role in evaluating and accepting the risks taken by the living donor. Some workers argue to set aside medical paternalism on behalf of the principle of donor autonomy. In this way the medical rule "primum non nocere" is overcome. Transplantation centers should reason beyond the shortage of organs and think in terms of the care for both donor and recipient.

Drosophila: a model for understanding obesity and diabetic complications.

The molecular mechanisms underlying development of obesity and diabetic complications are not well understood. Drosophila has become a popular model organism for studying a variety of human diseases. We discuss here emerging Drosophila models of obesity and diabetic complications.

Addition of infliximab compared with addition of sulfasalazine and hydroxychloroquine to methotrexate in patients with early rheumatoid arthritis (Swefot trial): 1-year results of a randomised trial.

BACKGROUND: New treatment strategies for early rheumatoid arthritis are evolving rapidly. We aimed to compare addition of conventional disease-modifying antirheumatic drugs (sulfasalazine and hydroxychloroquine) with addition of a tumour necrosis factor antagonist (infliximab) to methotrexate in patients with early rheumatoid arthritis. METHODS: We undertook a randomised trial in 15 rheumatology units in Sweden. We enrolled patients with early rheumatoid arthritis (symptom duration <1 year) and administered methotrexate (up to 20 mg per week). After 3-4 months, those who had not achieved low disease activity but who could tolerate methotrexate were randomly allocated by computer addition of either sulfasalazine and hydroxychloroquine or infliximab. Primary outcome was achievement of a good response according to European League Against Rheumatism (EULAR) criteria at 12 months. Patients were followed up to 24 months; here, we present findings at 12 months. Analysis was by intention to treat and we used non-responder imputation. The Swefot (Swedish Pharmacotherapy) study is registered in the WHO database at the Karolinska University Hospital, number CT20080004. FINDINGS: 487 patients were initially enrolled. Of 258 who had not achieved low disease activity with methotrexate, 130 were allocated sulfasalazine and hydroxychloroquine and 128 were assigned infliximab. 32 of 130 (25%) patients allocated sulfasalazine and hydroxychloroquine achieved the primary outcome compared with 50 of 128 (39%) assigned infliximab (risk ratio 1.59 [95% CI 1.10-2.30], p=0.0160). Adverse events were balanced fairly well between the two groups and accorded with known adverse events of the drugs used. No deaths occurred in either group. INTERPRETATION: In patients with early rheumatoid arthritis in whom methotrexate treatment failed, addition of a tumour necrosis factor antagonist to methotrexate monotherapy is clinically superior to addition of conventional disease-modifying antirheumatic drugs. FUNDING: Swedish Rheumatism Association, Schering-Plough.

Structural features of a water soluble gum polysaccharide from Murraya paniculata fruits.

A water soluble gum polysaccharide was isolated from Murraya paniculata fruits. Hydrolytic experiments, methylation analysis, periodate oxidation studies and NMR data revealed that the polysaccharide was extensively branched and it consisted of 1,3-, and 1,3,6-linked beta-D-galactopyranosyl units, terminal beta-D-galactopyranosyl units and terminal alpha-D-glucopyranosyl 1,4-beta-D-galactopyranosyl units. Small amounts of 4-O-methylglucuronic acid residues were also present.

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy.

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

Remission and response to early treatment of RA assessed by the Disease Activity Score. BARFOT study group. Better Anti-rheumatic Farmacotherapy.

OBJECTIVE: To assess criteria for individual response and remission based on the Disease Activity Score (DAS) in patients with RA participating in a long-term observational study. METHODS: The EULAR (European League against Rheumatism) criteria for individual response and a recently proposed remission criterion, DAS < 1.6, were applied to 90 patients with RA after treatment for 2 yr with disease-modifying anti-rheumatic drugs (DMARDs) and/or corticosteroids. RESULTS: Seventy-six per cent of the patients were classified as responders (46% good and 30% moderate responders). Good responders had significantly more improvement in pain and Health Assessment Questionnaire (HAQ) score than moderate responders and non-responders. Paired comparisons showed significant X-ray progression both for moderate responders and non-responders but not for good responders. Twenty-nine per cent of all responders had an end-point DAS > 2.4, indicating active disease. In this group of responders, X-ray changes progressed significantly, but this could not be demonstrated in the group of responders with DAS < or = 2.4. Thirty-six per cent of the patients included in the study were classified as being in remission after 2 yr of treatment. The group of patients in remission showed no evidence of X-ray progression after 2 yr. CONCLUSIONS: Response and remission criteria based on DAS were useful in a study of patients with RA who were managed essentially as in clinical practice. The criteria used showed construct and criterion validity, although discriminant validity could not be shown. The application of valid criteria for response and remission in clinical practice may be a useful aid in the evaluation of treatment effects and in making treatment decisions for individual patients.

Dpp gradient formation in the Drosophila wing imaginal disc.

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. Here we make use of a biologically active form of Dpp tagged with GFP to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusted to compartment size when this was experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation.

Structure of dicarboxyl malto-oligomers isolated from hypochlorite-oxidised potato starch studied by 1H and 13C NMR spectroscopy.

The main oxidised component in hypochlorite-oxidised potato starch was isolated by anion-exchange chromatography after enzymatic hydrolysis. The primary structure of the isolated oligosaccharides was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The isolated pentamer and hexamer contained one glucose unit oxidised to a dicarboxyl residue. As the hypochlorite oxidation has occurred at positions C-2 and C-3 of a glucose unit, the introduced carboxyl groups caused ring cleavage between the carbons C-2 and C-3. The ring-cleaved dicarboxyl residue had glycosidic linkages on both sides, implying that this oxidation pathway does not result in depolymerisation. The vicinal coupling constant between H-4 and H-5 in the ring-cleaved dicarboxyl residue was 3.2 Hz, showing that the gauche orientations are preferred. As a result, a different bending of the starch chain is observed and is probably, therefore, one of the reasons why hypochlorite oxidation reduces the tendency to retrogradation. The pKa values (3.0) were determined from the pH-dependent chemical shifts of H-1, H-4 and H-5 of the dicarboxylic residue.

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.

Activity of an Aspergillus terreus alpha-arabinofuranosidase on phenolic-substituted oligosaccharides.

The effect of phenolic substitutions on the activity of an alpha-arabinofuranosidase from Aspergillus terreus was investigated using feruloylated oligosaccharides isolated from plant cell walls, equivalent oligosaccharides obtained through treatment with specific ferulic acid esterases, and a synthetic lignin-carbohydrate complex (LCC). Feruloyl substituents limited the hydrolysis of arabinoxylan and arabinan oligosaccharides but only if the feruloyl group was esterified to the terminal non-reducing arabinose. Somewhat surprisingly, the LCC-model compound, in which the arabinose residue is substituted with a bulky dilignol group, was degraded by the enzyme. This indicated that the enzyme is able to approach this linkage from the xylose side.

Chromosome arrangement within a bacterium.

BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO. A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp.

The di-, tri- and tetrasaccharides formed during Trichoderma reesei endo-beta-D-mannanase treatment of pine kraft pulp were studied. The oligosaccharides in the hydrolysate were fractionated using size-exclusion, anion exchange and activated carbon chromatography. The primary sequence of the purified oligomers was determined by two-dimensional NMR techniques. The T. reesei mannanase cleaves the beta-1,4-glycosidic linkage of D-mannosyl residues attached either to D-mannose or D-glucose. The D-mannosyl residue may also be substituted by a D-galactosyl group. The main disaccharide produced was mannobiose, but a significant amount of 4-O-beta-D-glucopyranosyl-D-mannopyranose (GlcMan) was also produced. After extensive hydrolysis the main trisaccharides produced were 4-O-beta-D-mannopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1Man2) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-D-mannopyranose (Glc2Man). Some mannotriose 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyra-nosyl-D-manno pyranose (GlcMan2) and 4-O-beta-D-glucopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1GlcMan) were also detected in the hydrolysate. The structures of two tetrasaccharides were studied. They appeared to be 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-4-O-beta-D- glucopyranosyl-D-mannopyranose (Glc3Man) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyranosyl-4-O-beta-D -glucopyranosyl-D-mannopyranose (GlcManGlcMan). According to the results obtained, the galactoglucomannan in pine contains regions in which two or three glucose units are linked together, which further means that it may contain regions with several successive mannose residues. The galactose side groups were found to be attached only to mannose.

Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.

We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.