Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.     

Changes of Genotype,Sensitivity and Aggressiveness in Phytophthora infestans Isolates Collected in European Countries in 1997, 2006 and 2007

A total of 241 isolates of Phytophthora infestans were collected in 1997, 2006 and 2007 in eight European countries and characterized with molecular markers (simple sequence repeats, SSR genotypes) and phenotypic traits such as sensitivity to fungicides, mating type and aggressiveness. The mating type distribution changed from mainly A1 in 1997 to a majority of A2 in 2007. No resistant isolates were detected for fluazinam and mandipropamid, whereas the proportion of isolates resistant to mefenoxam (MFX) was high and increased over the years. There was no genetic link between mating type and MFX resistance. Aggressiveness (product between lesion expansion and sporulation capacity) was slightly higher for MFX‐resistant compared to sensitive isolates and for isolates collected later compared to earlier in the same season. It was about equally high for A1 and A2 types, and for French isolates in 1997 and British isolates in 2007, but lower for French isolates in 2007. Six different SSR genotype families were distinguished. In 1997, populations were dominated by genotype families I and III/IV, which significantly declined in 2007 being largely displaced by genotype families II (‘blue 13’ type) and V, which are by coincidence mainly A2 MFX resistant and A1 MFX sensitive, respectively. However, mating type and MFX resistance were genetically not linked to SSR genotypes.     

In ovo exposure to monochromatic green light promotes skeletal muscle cell proliferation and affects myofiber growth in posthatch chicks

Our previous studies demonstrated that illumination of chicken embryos with monochromatic green light results in enhanced body and muscle weight at later posthatch stages. In the present study, we investigated the cellular and molecular basis of this phenomenon. First, we showed that on day 6 posthatch, myofibers were more uniform in the in ovo illuminated group than in the control group incubated in the dark, with respect to the number of myofibers displaying diameter values within the range of the mean value. Second, we tested the hypothesis that in ovo illumination causes an increase in the number of myoblasts; this in turn can promote posthatch muscle growth. Indeed, a significant increase in the number of skeletal muscle cells isolated from pectoralis muscle was observed in the in ovo illuminated group on days 1 and 3 posthatch relative to the control group. This increased cell number was accompanied by higher expression levels of Pax7 and myogenin proteins on posthatch days 1 and 3, respectively. A parallel analysis of proliferating cells in the intact muscle further demonstrated a significant increase in the number of cells positive for proliferating cell nuclear antigen in muscle from the in ovo illuminated group. Third, we demonstrated that the transition from fetal- to adult-type myoblasts, normally occurring in late stages of chicken embryogenesis, is initiated earlier in embryos subjected to in ovo green-light illumination. We suggest that the stimulatory effect of in ovo illumination on posthatch muscle growth is the result of enhanced proliferation and differentiation of adult myoblasts and myofiber synchronization.     

A contemporary atlas of the mouse diaphragm: myogenicity, vascularity, and the pax3 connection

The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this "contemporary atlas" can be further explored in the context of diaphragm pathology and genetic disorders.     

Relation between augmentation index and adiponectin during one-year metformin treatment for nonalcoholic steatohepatosis: effects beyond glucose lowering?

ABSTRACT: BACKGROUND: Insulin resistance (IR) is the major driving force behind development and progression of atherosclerosis in patients with nonalcoholic fatty liver disease (NAFLD). Therefore, correction of IR is a relevant therapeutic target. We performed the current trial to evaluate whether 12- month metformin therapy improves vascular stiffness in patients with NAFLD and to assess if this improvement is associated with change in glucose control, insulin resistance or circulating adiponectin. METHODS: In randomized, placebo controlled study, 63 patients with NAFLD were assigned to one of two groups: Group 1 received daily metformin; Group 2 received placebo. Central aortic augmentation index (AI) was performed using SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia) at baseline, at 4-and 12-month treatment period. Metabolic parameters, insulin resistance markers and serum adiponectin levels were determined. RESULTS: In placebo group: AI did not improve during the treatment period. Liver function and adiponectin levels did not change during the study. In multiple linear regression analysis, the independent predictors of arterial stiffness improvement were metformin treatment and increase in circulating adiponectin levels. Among metformin treated patients: AI decreased significantly during the study. ALP and ALT decreased during initial 4-month treatment period, however raised to the pretreatment levels after 12 months. Serum adiponectin level tended to increase during treatment period with metformin. CONCLUSIONS: Metformin treatment was associated with significant decrease in AI during one year treatment in NAFLD patients. These beneficial vascular effects was associated with exposure to metformin per se as well as change in adiponectin levels suggesting that metformin may mediate its vascular effects via glicemic control-independent mechanisms.     

C-Met expression and mechanical activation of satellite cells on cultured muscle fibers.

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.     

alpha2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells

Placental protein 14 (PP14; glycodelin) is a pregnancy-associated immunoregulatory protein that is known to inhibit T cells via T-cell receptor desensitization. The recent demonstration of PP14 as lectin has provided insight into how it may mediate its CD45 glycoprotein-dependent T-cell inhibition. In this study, we have investigated PP14's lectin-binding properties in detail. Significantly, PP14 reacts with N-acetyllactosamine (LacNAc) as was also found for members of the galectin family, such as the potent immunoregulatory protein, galectin-1. However, in contrast to galectin-1, PP14's binding is significantly enhanced by alpha2,6-sialylation and also by the presence of cations. This was demonstrated by preferential binding to fetuin as compared with its desialylated variant asialofetuin (ASF) and by using free alpha2,6- versus alpha2,3-sialylated forms of LacNAc in competitive inhibition and direct solid-phase binding assays. Interestingly, from immunological point of view, PP14 also binds differentially to CD45 isoforms known to differ in their degree of sialylation. PP14 preferentially inhibits CD45RA+, as compared with CD45RO+ T cells, and preferentially co-capped this variant CD45 on the T-cell surface. Finally, we demonstrate that PP14 promotes CD45 dimerization and clustering, a phenomenon that may regulate CD45 activity.