Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SF1 DNA helicases, is discussed in the light of available structural and biochemical data.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

Heterozygote excess in a self-incompatible and partially clonal forest tree species -- Prunus avium L

Wild cherry (Prunus avium L.), a partially asexual self-incompatible forest tree, shows heterozygote excess, which is a poorly studied phenomenon. In three natural populations, we found significant heterozygote excess at almost all investigated loci (eight microsatellites and markers for the self-incompatibility locus). We examined four hypotheses to account for this observed heterozygote excess. First, negative F(IS) can result from a lack of selfed progeny in small populations of outcrossing species. A second explanation for negative F(IS) is selection during the life cycle of the most heterozygous individuals. A third explanation is negative assortative mating when reproduction occurs between individuals bearing phenotypes more dissimilar than by chance. The last explanation for negative F(IS) relies on asexual reproduction. Expectations for each hypothesis were tested using empirical data. Patterns of F(IS) differed among loci. Nevertheless, our experimental results did not confirm the small sample size hypothesis. Although one locus is probably under a hitch-hiking effect from the SI locus, we rejected the effect of the self-incompatibility locus for the genome as a whole. Similarly, although one locus showed a clear pattern consistent with the selection of heterozygous individuals, the heterosis effect over the whole genome was rejected. Finally, our results revealed that clonality probably explains significant negative F(IS) in wild cherry populations when considering all individuals. More theoretical effort is needed to develop expectations and hypotheses, and test them in the case of species combining self-incompatibility and partially asexual reproduction.     

IL-12 contributes to allergen-induced airway inflammation in experimental asthma

Lack of sufficient IL-12 production has been suggested to be one of the basic underlying mechanisms in atopy, but a potential role of IL-12 in established allergic airway disease remains unclear. We took advantage of a mouse model of experimental asthma to study the role of IL-12 during the development of bronchial inflammation. Administration of anti-IL-12p35 or anti-IL-12p40 mAb to previously OVA-sensitized BALB/c mice concomitantly with exposure to nebulized OVA, abolished both the development of bronchial hyperresponsiveness to metacholine as well as the eosinophilia in bronchoalveolar lavage fluid and peripheral blood. Anti-IL-12 treatment reduced CD4(+) T cell numbers and IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid and the mRNA expression of IL-10, eotaxin, RANTES, MCP-1, and VCAM-1 in the lung. Anti-IL-12p35 treatment failed to show these effects in IFN-gamma knockout mice pointing to the essential role of IFN-gamma in IL-12-induced effects. Neutralization of IL-12 during the sensitization process aggravated the subsequent development of allergic airway inflammation. These data together with recent information on the role of dendritic cells in both the sensitization and effector phase of allergic respiratory diseases demonstrate a dual role of IL-12. Whereas IL-12 counteracts Th2 sensitization, it contributes to full-blown allergic airway disease upon airway allergen exposure in the postsensitization phase, with enhanced recruitment of CD4(+) T cells and eosinophils and with up-regulation of Th2 cytokines, chemokines, and VCAM-1. IFN-gamma-producing cells or cells dependent on IFN-gamma activity, play a major role in this unexpected proinflammatory effect of IL-12 in allergic airway disease.     

A Large-Scale Distribution of Milk-Based Fortified Spreads: Evidence for a New Approach in Regions with High Burden of Acute Malnutrition

Background

There are 146 million underweight children in the developing world, which contribute to up to half of the world''s child deaths. In high burden regions for malnutrition, the treatment of individual children is limited by available resources. Here, we evaluate a large-scale distribution of a nutritional supplement on the prevention of wasting.

Methods and Findings

A new ready-to-use food (RUF) was developed as a diet supplement for children under three. The intervention consisted of six monthly distributions of RUF during the 2007 hunger gap in a district of Maradi region, Niger, for approximately 60,000 children (length: 60–85 cm). At each distribution, all children over 65 cm had their Mid-Upper Arm Circumference (MUAC) recorded. Admission trends for severe wasting (WFH<70% NCHS) in Maradi, 2002–2005 show an increase every year during the hunger gap. In contrast, in 2007, throughout the period of the distribution, the incidence of severe acute malnutrition (MUAC<110 mm) remained at extremely low levels. Comparison of year-over-year admissions to the therapeutic feeding program shows that the 2007 blanket distribution had essentially the same flattening effect on the seasonal rise in admissions as the 2006 individualized treatment of almost 60,000 children moderately wasted.

Conclusions

These results demonstrate the potential for distribution of fortified spreads to reduce the incidence of severe wasting in large population of children 6–36 months of age. Although further information is needed on the cost-effectiveness of such distributions, these results highlight the importance of re-evaluating current nutritional strategies and international recommendations for high burden areas of childhood malnutrition.     

Nitric oxide donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked

An outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair cycle. D1 also undergoes redox-dependent phosphorylation prior to its degradation. Although it has been suggested that phosphorylation modulates D1 metabolism, reversible D1 phosphorylation was reported not to be essential for PS II repair in Arabidopsis. Thus, the involvement of phosphorylation in D1 degradation is controversial. We show here that nitric oxide donors inhibit in vivo phosphorylation of the D1 protein in Spirodela without inhibiting degradation of the protein. Thus, D1 phosphorylation is not tightly linked to D1 degradation in the intact plant.