Study of the SRR1-gene family in Arabidopsis, yeast and mouse cells

Abstract: Light is a very important environmental cue for plants. In addition to the energy for photosynthesis, it also provides information that is essential for many processes including seed germination, seedlings development, neighbours detection or transition from the vegetative to the reproductive state. Plants evolved different photoreceptors, among which the phytochromes (PHY), which are red/far-red photoreceptors. This family is composed of 5 members in Arabidopsis thaliana, among which phyB plays the major role for detection of red light. Phytochromes are also able to reset the phase of the circadian clock, which is composed of a complicated network of genes able to produce rhythms of about 24 hours, even in constant conditions. SRR1 (Sensitivity to Red light Reduced) is a gene that was shown to act in the phyB pathway as well as in the circadian clock. It was proposed to play a role in the maintenance of rhythms of the core oscillator because of the circadian phenotype of the srr1 mutant in constant light and in constant darkness. In the present study, we present data confirming the role of SRR1 in the core oscillator. Moreover, we show that SRR1 levels are not limiting for circadian rhythms nor for light perception. We show that the protein levels, the sub-cellular localisation or the complex in which SRR1 is found are not regulated in a circadian manner. Orthologues of SRR1 exist in numerous eukaryotes, forming a new gene family. None of the members of this family have been described. Here, we present data suggesting that the mouse orthologue of SRR1 may not be required for oscillation of the circadian clock of mouse cells in culture. The yeast gene (called BER1 for Benomyl REsistant) was studied to understand the biochemical function of this gene family. Based on synthetic genetic screens, a role of Ber1 was inferred in microtubules dynamics, N-terminal acetylation of protein and proteasome biogenesis. The effect of Ber1 on microtubules was confirmed by the observation that the ber1Δ mutant is more resistant to microtubule-depolymerising drugs and microscopic examination of microtubules in ber 1 Δ mutants. Complementation assays of ber1 Δ mutants and srrl mutants failed to reveal any obvious functional conservation of the mouse, yeast and Arabidopsis orthologues. In conclusion, the SRR1 family might encode genes that either plays different roles in different organisms, or have similar biochemical function but are involved in diverse pathway. Résumé: La lumière est un des facteurs abiotiques les plus important pour les plantes. En plus de l'énergie fournie pour la photosynthèse, elle fourni également de l'information nécessaire pour différents processus comme la germination, le développement des jeunes plantules, la détection de plantes avoisinantes ou encore la transition entre le développement végétatif et reproductif. Plusieurs types de photorécepteurs sont apparus chez les plantes au cours de l'évolution, notamment les phytochromes (PHI, qui perçoivent la lumière rouge et rouge lointaine. Cette famille est composé de 5 membres chez Arabidopsis thaliana, parmi lesquels phyB est le principal récepteur pour la lumière rouge. Les phytochromes sont aussi utiles pour la synchronisation entre les cycles jour-nuit dus à la rotation de la terre et l'horloge circadienne. Cette dernière est composée d'un réseau compliqué qui permet la production de rythmes capables de perdurer même en conditions constantes. SRRI (Sensitivity to Red light Reduced) est un gène qui agit dans la voie de signalisation de phyB ainsi que dans l'horloge circadienne. Il a été proposé que SRRI joue un rôle dans la maintenance des rythmes de l'oscillateur principal à cause des phénotypes circadiens du mutant srrl observés en lumière et en obscurité continue. Dans ce travail, nous présentons des données confirmant le rôle de SRR1 dans l'oscillateur principal. Nous montrons que les niveaux d'expression de SRRI ne sont pas limitants pour les rythmes circadiens ou la perception de la lumière. Enfin, nous montrons que le niveau d'accumulation de la protéine, sa localisation subcellulaire ou encore la taille du complexe dans lequel SRRl est trouvé ne sont pas régulés de façon circadiennes. Des orthologues de SRRI existent chez de nombreux eucaryotes, formant une nouvelle famille de gènes. Aucun des membres de cette famille n'a été étudié avant ce travail. Nous présentons des données suggérant que l'orthologue de la souris n'est peut-être pas requis pour les oscillations de l'horloge circadienne de cellules de souris en culture. Le gène de la levure (appelé SERI pour Benomyl REsistant) a été étudié afin de mieux comprendre la fonction biochimique de cette famille de gène. Une analyse par crible synthétique léthal a révélé un rôle de Ber1 dans la dynamique des microtubules, l'acétylation des protéines en N-terminal et la biogenèse du protéasome. L'effet de Ber1 sur les microtubules a été confirmé par l'observation du mutant ber1 en présence de drogue capable de dépolymériser les microtubules. Celui-ci est plus résistant à ces drogues que le type sauvage. Des expériences de complémentation n'ont pas montré de conservation de la fonction entre SRRI et ses homologues de souris ou de levure. En conclusion, la famille SRRI code pour des gènes qui pourraient avoir soit des rôles différents selon les organismes, soit la même fonction biochimique mais qui serait utile pour des voies de signalisation différentes.

Critical role for the lactate transporter, monocarboxylate transporter 1 (mct1), in the regeneration of peripheral nerves

Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

Energy requirements during reproduction and reproductive effort in shrews (Soricidae)

The energy requirements of reproducing and non-reproducing females of three species of Crocidura (C. russula, C. viaria, C. olivieri), and two species of Sorex (S. coronatus, S. minutus) were measured. Members of these two genera show different rates of metabolism and reproductive strategies (extreme altriciality and larger litter size in Sorex). During pregnancy, the daily energy intake (on either an absolute or a mass-specific basis) remained close to the non-reproductive value in all species. The absolute energy intake increased strongly after parturition and was influenced by the litter size. Peak energy intake of lactating females was extremely high, typically between 100% and 200% above the non-reproductive requirements in the Crocidura and about 300% above the non-reproductive intake in the Sorex. The mass-specific daily energy intake was reduced during lactation in the three smaller species but not in C. viaria and C. olivieri. This decrease probably involves the different thermoregulatory abilities and/or basal rate of metabolism of the pups. Average reproductive effort was about 50%, in the Crocidura species and above 150% in the Sorex species. The higher effort in the latter is partly due to a larger litter size. But in addition, extreme altriciality in the Sorex leads to an earlier increase in the energy requirements and thus is an energetically more expensive reproductive mode. The present results support the hypothesis that a higher basal rate of metabolism is associated with a higher reproductive effort in shrews.

Is human lactation a particularly efficient process?

The recommended dietary allowances of many expert committees (UK DHSS 1979, FAO/WHO/UNU 1985, USA NRC 1989) have set out the extra energy requirements necessary to support lactation on the basis of an efficiency of 80 per cent for human milk production. The metabolic efficiency of milk synthesis can be derived from the measurements of resting energy expenditure in lactating women and in a matched control group of non-pregnant non-lactating women. The results of the present study in Gambian women, as well as a review of human studies on energy expenditure during lactation performed in different countries, suggest an efficiency of human milk synthesis greater than the value currently used by expert committees. We propose that an average figure of 95 per cent would be more appropriate to calculate the energy cost of human lactation.

Cost-effectiveness of temozolomide for the treatment of newly diagnosed glioblastoma multiforme: a report from the EORTC 26981/22981 NCI-C CE3 Intergroup Study.

BACKGROUND: The study aimed to compare the cost-effectiveness of concomitant and adjuvant temozolomide (TMZ) for the treatment of newly diagnosed glioblastoma multiforme versus initial radiotherapy alone from a public health care perspective. METHODS: The economic evaluation was performed alongside a randomized, multicenter, phase 3 trial. The primary endpoint of the trial was overall survival. Costs included all direct medical costs. Economic data were collected prospectively for a subgroup of 219 patients (38%). Unit costs for drugs, procedures, laboratory and imaging, radiotherapy, and hospital costs per day were collected from the official national reimbursement lists based on 2004. For the cost-effectiveness analysis, survival was expressed as 2.5 years restricted mean estimates. The incremental cost-effectiveness ratio (ICER) was constructed. Confidence intervals for the ICER were calculated using the Fieller method and bootstrapping. RESULTS: The difference in 2.5 years restricted mean survival between the treatment arms was 0.25 life-years and the ICER was euro37,361 per life-year gained with a 95% confidence interval (CI) ranging from euro19,544 to euro123,616. The area between the survival curves of the treatment arms suggests an increase of the overall survival gain for a longer follow-up. An extrapolation of the overall survival per treatment arm and imputation of costs for the extrapolated survival showed a substantial reduction in ICER. CONCLUSIONS: The ICER of euro37,361 per life-year gained is a conservative estimate. We concluded that despite the high TMZ acquisition costs, the costs per life-year gained are comparable to accepted first-line treatment with chemotherapy in patients with cancer.

Continuous versus single bolus enteral nutrition: comparison of energy metabolism in humans.

Continuous respiratory exchange measurements were performed on five women and five men for 1 h before and 6 h after the administration of a milkshake (53% carbohydrates, 30% lipid, and 17% protein energy) given either as a single bolus dose or continuously during 3 h using a nasogastric tube. The energy administered corresponded to 2.3 times the postabsorptive resting energy expenditure. Resting energy expenditure, respiratory quotient, plasma glucose, and insulin concentrations increased sooner and steeper, and plasma free fatty acids levels decreased earlier with the meal ingested as a single dose than with continuous administration. The magnitude of nutrient-induced thermogenesis was greater (P less than 0.01) with the single dose (means +/- SE, 10.0 +/- 0.6%) than with the continuous administration (8.1 +/- 0.5%). The overall (6 h) substrate balances were not significantly different between the two modes of administration. It is concluded that the mode of enteral nutrient administration influences the immediate thermogenic response as well as changes in respiratory quotient, glycemia, and insulinemia; however, the overall nutrient balance was not affected by the mode of enteral nutrient administration.

Abnormalities of fuel utilization as predisposing to the development of obesity in humans.

A number of recent investigations in man have demonstrated that a low ratio of fat to carbohydrate oxidation (i.e., a high respiratory quotient or RQ) was associated with actual and/or subsequent body weight gain in obese non-diabetic Pima Indians, in American men of various ages and in post-obese European women investigated shortly after the cessation of a hypocaloric diet. It is well known that numerous exogenous and endogenous factors influence the RQ at rest such as: the level of feeding (positive vs. negative energy balance), the composition of food eaten (high vs. low carbohydrate), the size of the glycogen stores, the amount of adipose tissue as well as genetic factors. It should be stressed that some nutritional situations can co-exist during which a low ratio of fat to carbohydrate is observed (i.e., a high RQ) despite weight loss. Furthermore, in most studies mentioned above, the low fat to carbohydrate oxidation ratio explains less than 10% of the variance in weight gain, suggesting that numerous additional factors also play a substantial role in the onset of weight gain. It is concluded that: 1) a low fat to carbohydrate oxidation ratio or an abnormal fat oxidation is difficult to define quantitatively since it is largely influenced by the energy level and the composition of the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and Resting State Functional Connectivity of the Subthalamic Nucleus: Identification of Motor STN Parts and the Hyperdirect Pathway.

Deep brain stimulation (DBS) for Parkinson's disease often alleviates the motor symptoms, but causes cognitive and emotional side effects in a substantial number of cases. Identification of the motor part of the subthalamic nucleus (STN) as part of the presurgical workup could minimize these adverse effects. In this study, we assessed the STN's connectivity to motor, associative, and limbic brain areas, based on structural and functional connectivity analysis of volunteer data. For the structural connectivity, we used streamline counts derived from HARDI fiber tracking. The resulting tracks supported the existence of the so-called "hyperdirect" pathway in humans. Furthermore, we determined the connectivity of each STN voxel with the motor cortical areas. Functional connectivity was calculated based on functional MRI, as the correlation of the signal within a given brain voxel with the signal in the STN. Also, the signal per STN voxel was explained in terms of the correlation with motor or limbic brain seed ROI areas. Both right and left STN ROIs appeared to be structurally and functionally connected to brain areas that are part of the motor, associative, and limbic circuit. Furthermore, this study enabled us to assess the level of segregation of the STN motor part, which is relevant for the planning of STN DBS procedures.

Predicted effects of small decreases in energy expenditure on weight gain in adult women.

Small daily positive energy imbalances of 200 to 800 kJ (about 50 to 200 kcal) due to reduced resting energy expenditure (REE), reduced diet-induced thermogenesis, or physical inactivity are believed to predispose to obesity. However, estimates of the magnitude of the weight gain often fail to account for concurrent changes in body composition and increases in maintenance energy requirements as weight increases and energy equilibrium is re-established. Using previously reported data on body composition and REE in women and the energy cost of tissue deposition, we used mathematical models to predict the theoretical effect of a persistent reduction in energy expenditure on long-term weight gain, assuming no adaptation in energy intake. The analyses indicate the following effects of a reduced level of energy expenditure in lean and obese women: (i) REE rises more slowly with increasing degrees of obesity due to a declining proportion of the more metabolically active fat-free mass; so, for the same positive energy balance, a significantly greater weight gain is expected for obese than for lean women before energy equilibrium is re-established; (ii) due to the greater energy density of adipose tissue, the time course of weight gain to achieve energy balance is longer for obese subjects: in general, this is approximately five years for lean and ten years for obese women; (iii) the magnitude of weight gain of lean women in response to a reduced energy expenditure of 200 to 800 kJ/day is only about 3 to 15 kg, amounts insufficient to explain severe obesity.

The effect of treatment with calcitonin on vertebral fracture rate in osteoporosis.

The efficacy of treatments for osteoporosis does not become evident when evaluated by fracture incidence (FI). Vertebral FI decreased in all controlled studies on calcitonin, but not significantly. Small sample sizes and short periods of treatment may have masked a possible therapeutic benefit, but longer, controlled studies with sodium fluoride or etidronate in larger groups of patients also failed to show a decrease in FI. The present analysis of nine published, therapeutic studies which indicate the FI per year and the initial prevalence of vertebral fractures, examines the question of whether the initial prevalence of fractures has an effect on the subsequent incidence of new fractures and whether the therapeutic effects have to be evaluated as a function of the initial prevalence of fractures. Bearing in mind the differences in roentgenological evaluation and in the size and quality of the various studies, the analysis revealed (1) that in the control groups there was a higher FI in patients with more than three vertebral fractures at baseline (estimated odds ratio (OR) = 49, p = 0.011); (2) that a similar trend, although not statistically significant, was observed in treated patients; (3) that the groups of control patients treated for more than 1 year showed in general an increase in FI beyond the first year and that the reverse was true in treated patients. In conclusion, failure to allow for the initial prevalence of vertebral fractures at the individual level in therapeutic trials of calcitonin to treat osteoporosis and prevent new fractures might have contributed to the absence of a demonstrable benefit of the treatment in those studies.

Data-based approach for developing a physical activity frequency questionnaire.

Measurement of total energy expenditure may be crucial to an understanding of the relation between physical activity and disease and in order to frame public health intervention. To devise a self-administered physical activity frequency questionnaire (PAFQ), the following data-based approach was used. A 24-hour recall was administered to a random sample of 919 adult residents of Geneva, Switzerland. The data obtained were used to establish the list of activities (and their median duration) that contributed to 95% of the energy expended, separately for men and women. Activities that were trivial for the whole sample but that contributed to > or = 10% of an individual's energy expenditure were also selected. The final PAFQ lists 70 activities or group of activities with their typical duration. About 20 minutes are required for respondents to indicate the number of days and the number of hours per day that they performed each activity. The PAFQ method was validated against a heart rate monitor, a more objective method. The total energy estimated by the PAFQ in 41 volunteers correlated well (r = 0.76) with estimates using a heart rate monitor. The authors conclude that the design of their self-administered physical activity frequency questionnaire based on data from 24-hour recall appeared to accurately estimate energy expenditure.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells.

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Parenteral nutrition in the intensive care unit: cautious use improves outcome.

Critical illness is characterised by nutritional and metabolic disorders, resulting in increased muscle catabolism, fat-free mass loss, and hyperglycaemia. The objective of the nutritional support is to limit fat-free mass loss, which has negative consequences on clinical outcome and recovery. Early enteral nutrition is recommended by current guidelines as the first choice feeding route in ICU patients. However, enteral nutrition alone is frequently associated with insufficient coverage of the energy requirements, and subsequently energy deficit is correlated to worsened clinical outcome. Controlled trials have demonstrated that, in case of failure or contraindications to full enteral nutrition, parenteral nutrition administration on top of insufficient enteral nutrition within the first four days after admission could improve the clinical outcome, and may attenuate fat-free mass loss. Parenteral nutrition is cautious if all-in-one solutions are used, glycaemia controlled, and overnutrition avoided. Conversely, the systematic use of parenteral nutrition in the ICU patients without clear indication is not recommended during the first 48 hours. Specific methods, such as thigh ultra-sound imaging, 3rd lumbar vertebra-targeted computerised tomography and bioimpedance electrical analysis, may be helpful in the future to monitor fat-free mass during the ICU stay. Clinical studies are warranted to demonstrate whether an optimal nutritional management during the ICU stay promotes muscle mass and function, the recovery after critical illness and reduces the overall costs.

Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones.

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

Housewife or working mum--each to her own? The relevance of societal factors in the association between social roles and alcohol use among mothers in 16 industrialized countries.

Aims To investigate whether differences in gender-income equity at country level explain national differences in the links between alcohol use, and the combination of motherhood and paid labour. Design Cross-sectional data in 16 established market economies participating in the Gender, Alcohol and Culture: An International Study (GenACIS) study. Setting Population surveys. Participants A total of 12 454 mothers (aged 25-49 years). Measurements Alcohol use was assessed as the quantity per drinking day. Paid labour, having a partner, gender-income ratio at country level and the interaction between individual and country characteristics were regressed on alcohol consumed per drinking day using multi-level modelling. Findings Mothers with a partner who were in paid labour reported consuming more alcohol on drinking days than partnered housewives. In countries with high gender-income equity, mothers with a partner who were in paid labour drank less alcohol per occasion, while alcohol use was higher among working partnered mothers living in countries with lower income equity. Conclusion In countries which facilitate working mothers, daily alcohol use decreases as female social roles increase; in contrast, in countries where there are fewer incentives for mothers to remain in work, the protective effect of being a working mother (with partner) on alcohol use is weaker. These data suggest that a country's investment in measures to improve the compatibility of motherhood and paid labour may reduce women's alcohol use.