Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow mesenchymal stem cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine exposure. They began spontaneous beating after 2 weeks, and expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure. These cells had several types of action potentials: sinus-node-like and ventricular-cell-like action potentials. The isoform of contractile protein genes indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. They expressed alpha 1A, alpha 1B, alpha 1D, beta 1, and beta 2 adrenergic and M1 and M2 muscarinic receptors. Stimulation with phenylephrine, isoproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with CGP20712A (beta 1-selective blocker). These findings indicated that cell transplantation therapy for the patients with heart failure might possibly be achieved using the regenerated cardiomyocytes from autologous bone marrow cells in the near future.     

Application of mesenchymal stem cells for the regeneration of cardiomyocyte and its use for cell transplantation therapy

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow mesenchymal stem cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine exposure. They began spontaneous beating after 2 weeks, and expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure. These cells had several types of action potentials; sinus node-like and ventricular cell-like action potentials. The isoform of contractile protein genes indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. They expressed alpha1A, alpha1B, alpha1D, beta1, and beta2 adrenergic and M1 and M2 muscarinic receptors. Stimulation with phenylephrine, isoproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with CGP20712A (beta1-selective blocker). These findings indicated that cell transplantation therapy for the patients with heart failure might possibly be achieved using the regenerated cardiomyocytes from autologous bone marrow cells in the near future.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996     

Developmental changes in ventricular myosin isoenzymes of the tammar wallaby

Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult wallaby ventricle showed three myosin isoenzymes, V₁, V₂ and V₃; western blots using specific anti-α-MyHC and anti-β-MyHC antibodies showed their MyHC compositions to be αα, αβ and ββ, respectively. Ventricular muscle in early pouch joeys expressed predominantly β-MyHC. Up to 200 days, the time of initial pouch exit, α-MyHC content was around 5%. Thereafter, there was a sharp increase of α-MyHC expression to 35% by 242 days of age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change, and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased α-MyHC expression empowers the heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones.     

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with β1–4, α1–6 and β1–6 glycosidic bond were synthesized; Galβ1–4GlcNH₂, Galα1–6GlcNH₂, Galα1–6GlcNAc, Galβ1–6GlcNH₂, Galβ1–4Galβ1–4GlcNH₂ and Galβ1–4Galβ1–4GlcNAc. Galα1–6GlcNH₂ (MelNH₂) and glucosamine (GlcNH₂) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH₂. Adding Galα1–6GlcNH₂ or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galα1–6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galα1–6GlcNH₂ were stained. The difference in the stainability of the K562 cells by Galα1–6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galα1–6GlcNH₂ and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galα1–6GlcNH₂ binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (γ) antibodies).     

Over-expressed estrogen receptor-α up-regulates hTNF-α gene expression and down-regulates β-catenin signaling activity to induce the apoptosis and inhibit proliferation of LoVo colon cancer cells

TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERα, ERβ, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERα and ERβ antagonist) or MPP (ERα antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERα and ERβ was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERα and ERβ was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10⁻⁵ M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERα-dependent pathway by binding to this receptor and not by increasing the expression of ER. (Mol Cell Biochem xxx: 1–9, 2005)     

Cerebellar localization of the NO-receptive soluble guanylyl cyclase subunits-α2/β1 in non-human primates

Nitric-oxide-sensitive guanylyl cyclase (NO-sGC) plays a pivotal role in many second messenger cascades. Neurotransmission- and neuropathology-related changes in NO-sGC have been suggested. However, the cellular localization of NO-sGC in primate brains, including humans, remains unknown. Biochemical evidence has linked the α₂-subunit of NO-sGC directly to neurotransmission in rodents. Here, we have used a recently characterized subunit-specific antibody for the localization of the α₂-subunit on sections from the cerebelli of the common marmoset (Callithrix jacchus; New World monkey) and macaque monkeys (Macaca mulatta, M. fascicularis; Old World monkeys). In contrast to the more ubiquitous cytoplasmic presence of subunit-β₁, the α₂-subunit is mainly confined to the somato-dendritic membrane including the spines of the Purkinje cells. Only limited colocalization with presynaptically localized synaptophysin has been seen under our staining conditions, indicating a higher abundance of subunit-α₂ at the postsynaptic site. This localization indicates that subunit-α₂ links NO-sGC to neurotransmission, whereas subunit-β₁ may act as a cytoplasmic regulator/activator by contributing to active heterodimer formation via translocation from the cytoplasm to the cell membrane. The last-mentioned action may be a prerequisite for generating nitric-oxide-dependent, subcellular, and postsynaptically localized cGMP signals along neuronal processes.This study was supported by the Deutsche Forschungsgemeinschaft.     

Physiological significance of P2X receptor-mediated vasoconstriction in five different types of arteries in rats

P2X₁ receptors, the major subtype of P2X receptors in the vascular smooth muscle, are essential for α,β-methylene adenosine 5′-triphosphate (α,β-MeATP)-induced vasoconstriction. However, relative physiological significance of P2X₁ receptor-regulated vasoconstriction in the different types of arteries in the rat is not clear as compared with α₁-adrenoceptor-regulated vasoconstriction. In the present study, we found that vasoconstrictive responses to noncumulative administration of α,β-MeATP in the rat isolated mesenteric arteries were significantly smaller than those to single concentration administration of α,β-MeATP. Therefore, we firstly reported the characteristic of α,β-MeATP-regulated vasoconstrictions in rat tail, internal carotid, pulmonary, mesenteric arteries, and aorta using single concentration administration of α,β-MeATP. The rank order of maximal vasoconstrictions for α,β-MeATP (E _{max·α,β-MeATP}) was the same as that of maximal vasoconstrictions for noradrenaline (E _max·NA) in the internal carotid, pulmonary, mesenteric arteries, and aorta. Moreover, the value of (E _{max·α,β-MeATP}/E _max·KCl)/(E _max·NA/E _max·KCl) was 0.4 in each of the four arteries, but it was 0.8 in the tail artery. In conclusion, P2X₁ receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries, and aorta, but much greater in the tail artery, suggesting its special role in physiological function.     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

The voltage-gated calcium-channel β subunit: more than just an accessory

Voltage-gated Ca²⁺ channels (VGCCs) are involved in a number of excitatory processes in the cell that regulate muscle contraction, neurotransmitter release, gene regulation, and neuronal migration. They consist of a central pore-forming α₁ subunit together with a number of associated auxiliary subunits including a cytoplasmic β subunit. With the aid of X-ray crystallography, it has been found that the β subunits of VGCCs (β_2a, β₃, and β₄) interact strongly with the I–II loop of the pore-forming α₁ subunit. Here we discuss the potential interaction sites of β_1a with its α₁ subunit as well as the skeletal ryanodine receptor. We suggest that not only can β_1a interact with the α₁ subunit I–II loop, but more subtle interactions may be possible through the II–III loop via the β_1a SH3 domain. Such findings could have important implications with respect to EC coupling.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

MHC class I genes of the channel catfish: sequence analysis and expression

 Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β₂-microglobulin (β₂m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 M _r glycoprotein (presumably class I α) was associated with β₂m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β₂m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts. Received: 25 March 1998 / Revised: 28 July 1998     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Comparative C19-radiosteroid metabolism by MA 160 and HeLa cell lines

Summary Since the designation of the human MA 160 line as prostatic epithelial cells has been questioned and the possibility of HeLa cross contamination raised, this comparative study of C₁₉-radiosteroid transformation in MA 160 and HeLa monolayer cultures was done to determine whether these cells possess the distinguishing features of reductive and oxidative androgen metabolism expected in male and female genital organs, respectively. We compared the radiometabolite patterns produced by incubating [¹⁴C]testosterone (300nM) and [³H]testosterone (3nm) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) with cultures of prostatic MA 160 and HeLa Parent, TCRC-1, TCRC-2 and ATC 229 cells. C₁₉-Radiosteroid metabolite patterns from MA 160 cell incubations also were compared with patterns generated by MA 196 fibroblasts from abdomnal skin of the same donor. MA 160 cells metabolized radiotestosterone predominantly to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The diol epimers were the principal metabolites of 5α-dihydrotestosterone radiosubstrate. In contrast, radiotestosterone metabolism by MA 196 and HeLa Parent, TCRC-1 and TCRC-2 cells was overwhelmingly to the 17-oxosteroids 4-androstene-3,17-dione and androsterone. Another pathway was operative in HeLa 229 and, to a minor extent, in TCRC-1, which converted radiotestosterone to 4-androstene-3α,17β-diol and 5α-androstane-3α,17β-dol, with little formation of 5α-dihydrotestosterone. MA 160 cells thus metabolize radiotestosterone preponderantly to 5α-reduced 17β-hydroxysteroids as expected for prostatic epithelial cells, whereas HeLa cells show heterogeneity in metabolizing the labeled hormone by the alternative 17-oxosteroid and Δ⁴ pathways. This work was supported by Public Health Service Research Grants CA 13417 and CA 12924 from the National Cancer Institute, AM 11011 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and by appropriations of the Commonwealth of Massachusetts, Item No. 4532-9003-01.     

Heterogeneity of α-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V_1a, V_1b,V_1c, V₂ and V₃, in order of decreasing mobility. In western blots, V_1a, V_1b, and V_1c reacted with anti-α-MyHC antibody, but not with anti-β-MyHC, whereas V₂ and V₃ reacted with anti-β-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-α-MyHC antibody while the third bound anti-β-MyHC antibody. We conclude that V_1a, V_1b, V_1c are triplets arising from the dimerization of two distinct α-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased α-MyHC content significantly (t-test, P < 0.001) from 91.6 ± 5.9% (SEM, n = 4) in control animals to 67.2 ± 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in β-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.     

Interactions between tachykinins and diverse,human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured using⁸⁶Rb⁺ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC₅₀∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC₅₀∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC₅₀∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC₅₀ values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [³H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ_1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ_1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, α_S- and β-casein. Likewise, incubating Aβ_1–40 with αB-crystallin inhibited both Aβ_1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.     

Effects of uniaxial cyclic strain on adipose-derived stem cell morphology,proliferation, and differentiation

Cells and tissues in vivo are subjected to various forms of mechanical forces that are essential to their normal development and functions. The arterial blood vessel wall is continuously exposed to mechanical stresses such as pressure, strain, and shear due to the pulsatile nature of blood flow. Vascular smooth muscle cells (SMCs) populate the media of blood vessels and play important roles in the control of vasoactivity and the remodeling of the vessel wall. It is well documented that the phenotype and functions of vascular SMCs are not only regulated by chemical factors such as transforming growth factor-β₁ (TGF-β₁), but also by mechanical factors such as uniaxial strain. The purpose of our study was to explore the effects of TGF-β₁ alone or in combination with uniaxial cyclic strain on adipose-derived stem cell (ASC) morphology, proliferation, and differentiation. Low passage ASCs were stimulated with 10% strain at 1 Hz for 7 days, with or without TGF-β₁. Cyclic strain inhibited proliferation, and caused alignment of the cells and of the F-actin cytoskeleton perpendicular to the direction of strain. Strain alone resulted in a decrease in the expression of early SMC markers α-SMA and h ₁-calponin. While the response of SMCs and other progenitor cells such as bone marrow stromal cells to mechanical forces has been extensively studied, the roles of these forces on ASCs remain unexplored. This work advances our understanding of the mechanical regulation of ASCs. Presented in part at the third annual meeting of the International Fat Applied Technology Society (IFATS), September 10–13, 2005, Omni Charlottesville Hotel, Charlottesville, VA, USA.