Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

Cloning and characterization of a phospholipase C from the C(4) plant Digitaria sanguinalis

As a PLC activity was implicated in the light transduction pathway that controls C(4) photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned. The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca(2+) concentration, and with different sensitivity to the PLC inhibitor U-73122.     

Fractionation analysis of the endosomal compartment during rat reticulocyte maturation

A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10-40%). Two fractions were isolated: P1 at the 25-35% sucrose interface and P2 at the 17-25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA.     

Physiological adaptations to exercise in people with spinal cord injury

The number of patients that suffer some type of spinal cord lesion in recent years are high and have increased because of factors such as traffic accidents. Although their life expectancy has increased, cardiovascular illnesses is one of the main causes of morbidity and mortality. Since the degree of physical fitness is an important factor regarding the risk of cardiovascular disease, the objective of the present study was to examine the global adaptation (cardiorespiratory, metabolic and thermoregulatory response) of the organism to exercise and the application of this data to the habitual practice of physical activity to improve state of health. A group of 42 patients with spinal injury, 85% of whom were paraplegic and the remaining 15% tetraplegic performed 42 exercise tests on a cycloergometer. Body temperature (tympanum, surface of the deltoids and surface of the back), metabolic parameters (plasma uric acid, glycemia, plasma lactate), cardiocirculatory adaptation (heart rate, blood pressure arm, blood pressure leg) and ventilatory adaptation (VO2, VCO2, fr Vt, VE) were monitored. Blood pressure in the arm, blood concentrations of lactate and ventilatory parameters showed an evolution statistically dependent on the work to which the subject was submitted. Heart rate showed a statistically significant correlation with the ventilatory parameters and work load. The proportional response of the cardioventilatory parameters to the increase in the work load allowed us to evaluate the repercussion of a given exercise and thus avoid exercise of an excessive intensity that could produce cardiocirculatory changes that might entail an added risk. Heart rate presents an excellent correlation, shown in this work, with the oxygen consumption and could therefore be used to quantify the cardiorespiratory and metabolic repercussion of the exercise carried out. Furthermore, this quantification may allow for the adaptation of exercise intensity to the patient thus improving the results obtained from the practice of exercise that has been proven so necessary in these patients.     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

Characterization of salt stress-enhanced phosphoenolpyruvate carboxylase kinase activity in leaves of <Emphasis Type="Italic">Sorghum</Emphasis><Emphasis Type="Italic">vulgare</Emphasis>: independence from osmotic stress,involvement of ion toxicity and significance of dark phosphorylation

C(4) phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) is subjected to in vivo regulatory phosphorylation by a light up-regulated, calcium-independent protein kinase. Salt stress greatly enhanced phosphoenolpyruvate carboxylase-kinase (PEPCase-k) activity in leaves of Sorghum. The increase in PEPCase-k anticipated the time course of proline accumulation thereby suggesting that water stress was not involved in the kinase response to salt. Moreover, osmotic stress seemed not to be the main factor implicated, as demonstrated by the lack of effect when water availability was restricted by mannitol. In contrast, LiCl (at a concentration of 10 mM in short-term treatment of both excised leaves and whole plants) mimicked the effects of 172 mM NaCl salt-acclimation, indicating that the rise in PEPCase-k activity resulted primarily from the ionic stress. Both NaCl and LiCl treatments increased the activity of a Ca(2+)-independent, 35 kDa kinase, as demonstrated by an in-gel phosphorylation experiment. Short-term treatment of excised leaves with NaCl or LiCl partially reproduces the effects of whole plant treatments. Finally, salinization also increased PEPCase-k activity and the phosphorylation state of PEPCase in darkened Sorghum leaves. This fact, together with increased malate production during the dark period, suggests a shift towards mixed C(4) and crassulacean acid metabolism types of photosynthesis in response to salt stress.     

Integrating 'omic' information: a bridge between genomics and systems biology

The availability of genome sequences for several organisms, including humans, and the resulting first-approximation lists of genes, have allowed a transition from molecular biology to 'modular biology'. In modular biology, biological processes of interest, or modules, are studied as complex systems of functionally interacting macromolecules. Functional genomic and proteomic ('omic') approaches can be helpful to accelerate the identification of the genes and gene products involved in particular modules, and to describe the functional relationships between them. However, the data emerging from individual omic approaches should be viewed with caution because of the occurrence of false-negative and false-positive results and because single annotations are not sufficient for an understanding of gene function. To increase the reliability of gene function annotation, multiple independent datasets need to be integrated. Here, we review the recent development of strategies for such integration and we argue that these will be important for a systems approach to modular biology.