Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

Restoration of Responsiveness of Phospholipase Cγ2-Deficient Platelets by Enforced Expression of Phospholipase Cγ1

Receptor-mediated platelet activation requires phospholipase C (PLC) activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT), PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbβ3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbβ3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistently, αIIbβ3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbβ3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbβ3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2.     

Total synthesis and biological evaluation of novel C2–C6 region analogues of dictyostatin

By exploiting a Still–Gennari HWE coupling with a common C11–C26 aldehyde, a series of C2–C6 modified analogues of the microtubule-stabilising marine natural product dictyostatin were synthesised and evaluated in vitro for growth inhibition against a range of human cancer cell lines, including the (P-glycoprotein efflux-mediated) Taxol-resistant NCI/ADR cell line. Removal of the C6 methyl substituent in dictyostatin was found to be well tolerated and led to the retention of antiproliferative activity in the low nanomolar range (IC₅₀ = 43 nM in the NCI/ADR cell line), while partial and full saturation of the (2Z, 4E)-dienoate region led to a progressive reduction in biological potency. The lactone ring size was found to be critical, as C21 to C19 translactonisation to afford 20-membered isodictyostatin analogues led to a significant loss of cytotoxicity. In a series of incubatory experiments performed on the PANC-1 cell line, all three of the 22-membered macrolide analogues acted in an analogous fashion to dictyostatin, through a mechanism of microtubule stabilization, causing both an accumulation of cells at the G2/M phase and formation of characteristic dense intracellular microtubule bundles.     

Phylodynamics of hepatitis C virus subtype 2c in the province of Córdoba, Argentina

The Hepatitis C Virus Genotype 2 subtype 2c (HCV-2c) is detected as a low prevalence subtype in many countries, except in Southern Europe and Western Africa. The current epidemiology of HCV in Argentina, a low-prevalence country, shows the expected low prevalence for this subtype. However, this subtype is the most prevalent in the central province of Córdoba. Cruz del Eje (CdE), a small rural city of this province, shows a prevalence for HCV infections of 5%, being 90% of the samples classified as HCV-2c. In other locations of Córdoba Province (OLC) with lower prevalence for HCV, HCV-2c was recorded in about 50% of the samples. The phylogenetic analysis of samples from Córdoba Province consistently conformed a monophyletic group with HCV-2c sequences from all the countries where HCV-2c has been sequenced. The phylogeographic analysis showed an overall association between geographical traits and phylogeny, being these associations significant (α = 0.05) for Italy, France, Argentina (places other than Córdoba), Martinique, CdE and OLC. The coalescence analysis for samples from CdE, OLC and France yielded a Time for the Most Common Recent Ancestor of about 140 years, whereas its demographic reconstruction showed a “lag” phase in the viral population until 1880 and then an exponential growth until 1940. These results were also obtained when each geographical area was analyzed separately, suggesting that HCV-2c came into Córdoba province during the migration process, mainly from Europe, which is compatible with the history of Argentina of the early 20th century. This also suggests that the spread of HCV-2c occurred in Europe and South America almost simultaneously, possibly as a result of the advances in medicine technology of the first half of the 20th century.     

Analysis of δ13C of CO2 distinguishes between microbial respiration of added C4-sucrose and other soil respiration in a C3-ecosystem

The main aim of this study was to test various hypotheses regarding the changes in ¹³C of emitted CO₂ that follow the addition of C₄-sucrose to the soil of a C₃-ecosystem. It forms part of an experimental series designed to assess whether or not the contributions from C₃-respiration (root and microbial) and C₄-respiration (microbial) to total soil respiration can be calculated from such changes. A series of five experiments, three on sieved (root-free) mor-layer material, and two in the field with intact mor-layer (and consequently with active roots), were performed. Both in the experiments on sieved mor-layer and the field experiments, we found a C₄-sucrose-induced increase in C₃-respiration that accounted for between 30% and 40% of the respiration increase 1 h after sucrose addition. When the course of C₃-, C₄- and total respiration was followed in sieved material over four days following addition of C₄-sucrose, the initially increased respiration of C₃-C was transient, passing within less than 24 h. In a separate pot experiment, neither ectomycorrhizal Pinus sylvestrisL. roots nor non-mycorrhizal roots of this species showed respiratory changes in response to exogenous sucrose. No shift in the ¹³C of the evolved CO₂ after adding C₃-sucrose to sieved mor-layer material was found, confirming that the sucrose-induced increase in respiration of endogenous C was not an artefact of discrimination against ¹³C during respiration. Furthermore, we conclude that the C₄-sucrose induced transient increase in C₃-respiration is most likely the result of accelerated turnover of C in the microbial biomass. Thus, neither respiration of mycorrhizal roots, nor processes discriminating against ¹³C were likely sources of error in the field. The estimated ¹³C of evolved soil CO₂ in three field experiments lay between –25.2 and –23.6. The study shows that we can distinguish between CO₂ evolved from microbial mineralisation of added C₄-sucrose, and CO₂ evolved from endogenous carbon sources (roots and microbial respiration).     

Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 – A novel target of Runx2

Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2^Dox sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (?84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells.     

Interspecific variation in assimilation of 14CO2 into C4 acids by leaves of C3, C4 and C3−C4 intermediate Flaveria species near the CO2 compensation concentration

The assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of C₃, C₄ and C₃–C₄ intermediate Flaveria species was investigated near the CO₂ compensation concentration ^* in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO₂, the proportion of CO₂ fixed by PEP carboxylase at ^* increased in all six C₃–C₄ intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO₂, with only about a 1.6-fold enhancement of CO₂ assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C₃–C₄ species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C₃ species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at ^*. Assimilation of CO₂ by PEP carboxylase in the C₄ species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C₄-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO₂. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C₄-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with ¹²CO₂ for 5 min at ^* resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C₄ acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO₂ fixation by PEP carboxylase at the CO₂ compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related ¹⁴CO₂-labeling studies at air-levels of CO₂ which indicated that F. floridana and F. ramosissima were more C₄-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C₄ plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CO₂ compensation concentration - ^* a subatmospheric level of CO₂ approximating Published as Paper No. 8832, Journal Series, Nebraska Agricultural Research Division     

The 13C/12C isotopic signal of day-respired CO2 in variegated leaves of Pelargonium × hortorum

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.     

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca²⁺. Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca²⁺-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca²⁺-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.     

C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: Relevance to disease and ageing?

Reduced muscle mass and increased susceptibility to TNF‐induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C₂ myoblasts display diminished differentiation and increased susceptibility to TNF‐α‐induced cell death versus subcloned C₂C₁₂ cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan‐blue) and anabolic/catabolic parameters (RT‐PCR) over 72 h ± TNF‐α (20 ng ml^?1). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 ± 6.82 vs. 30.71 ± 2.5, P < 0.05; 544.94 ± 27.7 vs. 39.4 ± 3.37 mU mg ml^?1, P < 0.05), fold increases in myoD (21.45 ± 3.12 vs. 3.97 ± 1.76, P < 0.05; 31.07 ± 3.1 vs. 6.82 ± 1.93, P < 0.05) and myogenin mRNA (241.8 ± 40 vs. 36.80 ± 19.3, P < 0.05; 440 ± 100.5 vs. 201.1 ± 86, P < 0.05) were detected in C₂C₁₂ versus C₂. C₂C₁₂ showed significant increases in IGF‐I mRNA (243.05 ± 3.87 vs. 105.75 ± 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 ± 0.28 vs. 1.79 ± 0.29 mg ml^?1, P < 0.05) at 72 h versus C₂ cells. Significant temporal reductions in C₂C₁₂ IGFBP2 mRNA (28.02 ± 15.44, 13.82 ± 8.07, 6.92 ± 4.37, P < 0.05) contrasted increases in C₂s (4.31 ± 3.31, 13.02 ± 9.92, 82.9 ± 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF‐α increased cell death in C₂s (2.67 ± 1.54%, 34.42 ± 5.39%, 29.71 ± 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C₂C₁₂s at 48 h but caused a small significant increase at 72 h (9.88 ± 4.02% (TNF‐α) vs. 6.17 ± 0.749% (DM), 72 h). TNF‐α and TNFRI mRNA were unchanged; however, larger reductions in IGF‐I (8.2‐ and 7.5‐fold vs. 4.5‐ and 4.1‐fold (48, 72 h)), IGF‐IR (2‐fold vs. no‐significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C₂ versus C₂C₁₂ with TNF‐α. This investigation provides insight into regulators of altered basal hypertrophy and TNF‐induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders. J. Cell. Physiol. 225: 240–250, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C ε)

We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.     

A model of photosynthetic CO2 assimilation and carbon-isotope discrimination in leaves of certain C3−C4 intermediates

A model of leaf, photosynthesis has been developed for C₃–C₄ intermediate species found in the generaPanicum, Moricandia, Parthenium andMollugo where no functional C₄ pathway has been identified. Model assumptions are a functional C₃ cycle in both mesophyll and bundle-sheath cells and that glycine formed in the mesophyll, as a consequence of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39), diffuses to the bundle sheath, where most of the photorespiratory CO₂ is released. The model describes the observed gas-exchange characteristics of these C₃–C₄ intermediates, such as low CO₂-compensation points () at an O₂ pressure of 200 mbar, a curvilinear response of to changing O₂ pressures, and typical responses of CO₂-assimilation rate to intercellular CO₂ pressure. The model predicts that bundle-sheath CO₂ concentration is highest at low mesophyll CO₂ pressures and decreases as mesophyll CO₂ pressure increases. A partitioning of 5–15% of the total leaf Rubisco into the bundle-sheath cells and a bundlesheath conductance similar to that proposed for C₄ species best mimics the gas-exchange results. The model predicts C₃-like carbon-isotope discrimination for photosynthesis at atmospheric levels of CO₂, but at low CO₂ pressures it predicts a higher discrimination than is typically found during C₃ photosynthesis at lower CO₂ pressures.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate - p(CO₂) partial pressure of CO₂ - p(O₂) partial pressure of O₂. See also p. 471     

Monte Carlo simulation of the adsorption of C2–C7 linear alkanes in aluminophosphate AlPO4-11

The adsorption behaviors of linear alkanes ranging in length from C₂ to C₇ in AlPO₄-11 have been simulated by using configurational-bias Monte Carlo technique at 313 K. The calculated heats of adsorption at zero coverage for linear alkanes, estimated by Henry coefficients, are consistent well with previously reported experimental and simulation results. The simulated isotherms for n-hexane in AlPO₄-11 at 298 K agree with the experimental data. The isotherms of C₂–C₇ linear alkane were predicted, in which butane presents a substep. The adsorbed alkane molecules are only localized in 10-membered ring channels, and adsorbed phase structures for each alkane were investigated. Total potentials for individual alkane molecule decrease with increasing number of carbon atoms. A linear change in total potential is observed for each linear alkane with increasing loading per unit cell, except that an increasing trend is found in the total potential curve of butane as the loading per unit cell is higher than two molecules.     

N2(C2H2−C2H4) fixation in two species of Ceanothus seedlings in second year postfire chaparral

Nitrogen fixation in excised root nodules of 2-year-old, postfireCeanothus tomentosus andC. leucodermis seedlings was measured over an 8-month period using the acetylene reduction method. High levels of NO₃–N and NH₄–N present in postfire soils were limited to the upper 10 cm and did not inhibit nodulation in these deeper-rooting seedlings. Decreases in acetylene reduction activity occurred with decreased soil moisture and increased soil temperature. Nitrogen gains from these two Ceanothus shrub seedlings totalled 1.6 kg N ha^–1 yr^–1.     

Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and β-hydroxybutyrate

Pseudomonas AM1 grows on beta-hydroxybutyrate and methanol at similar rates. beta-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C(1) compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.     

C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

Synthesis and evaluation of C2 functionalized analogs of the α-tubulin-binding natural product pironetin

Pironetin is an α-tubulin-binding natural product with potent antiproliferative activity against several cancer cell lines that inhibits cell division by forming a covalent adduct with α-tubulin via a Michael addition into the natural product’s α,β-unsaturated lactone. We designed and prepared analogs carrying electron-withdrawing groups at the α-position (C2) of the α,β-unsaturated lactone with the goal to generate potent and selective binding analogs. We prepared derivatives containing halogens, a phenyl, and a methyl group at the C2 position to evaluate the structure-activity relationship at this position. Testing of the analogs in ovarian cancer cell lines demonstrated 100–1000-fold decreased antiproliferative activity.