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At least 523 records · Page 29Linked to original sources
PubMed2004

Expression and function of Tec, Itk, and Btk in lymphocytes: evidence for a unique role for Tec.

The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling, because mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. In this study, we show that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, we find that Tec overexpression in lymphocyte cell lines is sufficient to induce phospholipase Cgamma (PLC-gamma) phosphorylation and NFAT (nuclear factor of activated T cells) activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-gamma, but not the adapters LAT, SLP-76, and BLNK, which are required for Btk and Itk to couple to PLC-gamma. Finally, we show that the unique effector function for Tec correlates with a unique subcellular localization. We hypothesize that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed.

The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.

Amino Acid Sequence↗
PubMed2005

Effect of post-mating GnRH analogue (buserelin) treatment on PGF2alpha release in ewes and ewe lambs.

The objectives of this study were to determine the effects of buserelin or saline treatment on ovarian function (Experiment 1), plasma PGFM concentrations and oxytocin stimulated prostaglandin F(2alpha) (PGF(2alpha)) release (Experiment 2) in ewe lambs and ewes. Welsh Halfbred ewes (n=26) and ewe lambs (n=24) were mated to vasectomised rams at synchronised oestrus and on Day 12 post-mating each animal was injected intramuscularly either normal saline or 4 microg buserelin. In Experiment 1, plasma progesterone and oestradiol concentrations were determined in samples collected by jugular venepuncture 1h before and at 0, 2, 4, 6, 8, 24, 48 and 72 h after treatment (n=7 per treatment group). Progesterone concentrations increased (P<0.05) from 2 to 8h after buserelin treatment and returned to basal levels after 72 h, whereas oestradiol concentrations were maximal at 2h post-treatment and returned to basal levels after 24h (P<0.05). Oestradiol concentrations were lower (P<0.05) in buserelin-treated animals than controls at 72 h post-treatment. Basal and post-treatment progesterone concentrations were greater (P<0.05) in ewes than in ewe lambs but oestradiol levels were similar for both age groups. Ovulation rate, determined by laparoscopy on Day 14, was similar for both age groups (ewes 1.1; ewe lambs 1.0). Buserelin treatment induced accessory corpora lutea in ewes (4/7; 57%) but not in ewe lambs (0/7; 0%). In the Experiment 2, plasma PGFM concentrations were determined in samples collected at 20-min intervals for 6h on Day 14 and at 20-min intervals for 1h before and at 10-min intervals for 1h and then at 20-min intervals for a further 3h period after an intravenous injection of oxytocin (1IU/kg body weight) on Day 15 post-oestrus. In this experiment there were five ewe lambs and six ewes per treatment group. There was no effect of buserelin treatment or age on basal PGFM concentrations on either Day 14 or 15. Although peak PGFM concentrations tended to be lower in buserelin-treated animals, the difference was not significant (P>0.05). However, peak duration following oxytocin challenge on Day 15 post-mating was shorter (P<0.05) in control ewes compared with control ewe lambs. In conclusion, buserelin treatment given on Day 12 post-oestrus enhances luteal function more in ewes than ewe lambs and after a transitory increase, reduces oestradiol concentrations in both ewes and ewe lambs. However, buserelin treatment does not significantly attenuate the luteolytic signal.

Age Factors↗
PubMed1994

SK-N-BE: a human neuroblastoma cell line containing two subtypes of delta-opioid receptors.

A human neuroblastoma cell line, SK-N-BE, was shown to express a substantial amount of opioid receptors (200-300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of delta-opioid receptors. Results from sucrose-gradient sedimentation experiments were compared with similar data obtained with the mu-opioid receptor of the rabbit cerebellum and the delta-opioid receptor of the hybrid NG108-15 cell line and have shown that the opioid receptor of the SK-N-BE cell line behaved hydrodynamically as an intermediate between mu- and delta-opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK-N-BE cell membranes could belong to two delta-opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of delta-opioid receptor is negatively coupled to the adenylate cyclase via a Gi protein and that the opiate receptors of the SK-N-BE neuroblastoma cell line undergo a rapid down-regulation when preincubated in the presence of the high-affinity opioid, etorphine.

Adenylyl Cyclases↗
PubMed2001

Human and rat alveolar macrophages express multiple EDG receptors.

Endothelial differentiation gene (EDG) receptors are a new family of eight G protein-coupled receptors for the lysophospholipids lysophosphatitic acid and sphingosine-1-phosphate. In the present experiments, the expression of EDG receptors in rat and human alveolar macrophages was studied by reverse transcription-polymerase chain reaction (RT-PCR). In alveolar macrophages of both species, mRNA for multiple EDG receptors could be detected, but the pattern of expression was different in both species. In human alveolar macrophages, mRNA for EDG1, EDG2, EDG4, EDG7 receptors and, to a lesser extent, for the EDG7 receptor was detected, whereas in rat macrophages, mRNA for EDG2, EDG5 receptors and, to a lesser extent, for the EDG6 receptor was found. In functional experiments, it was observed that lysophosphatitic acid and sphingosine-1-phosphate can stimulate O(2)(-) generation in rat and human alveolar macrophages suggesting that lysophosphatitic acid and sphingosine-1-phosphate possibly acting via EDG receptors may play a role in controlling the activation of macrophages.

Animals↗
PubMed2002

Immunological effects of yogurt addition to a re-nutrition diet in a malnutrition experimental model.

The therapeutic and preventive effects of yogurt and lactic acid bacteria on diseases such as cancer, infection and gastrointestinal disorders are well ocumented. The aim of this research was to study the effects of different doses of yogurt addition after milk re-nutrition diet, on the recovery of the intestinal barrier and mucosal immune function. Experiments were performed on groups of mice, malnourished and re-nourished with milk during 7 d, and mice with diet supplemented with yogurt for 2, 5 and 7 consecutive d. Nutritional parameters such as weight gain, serum total protein, and the number of IgA, IgM and IgG B cells of the small intestine were determined. We also quantified intraepithelial leukocytes, mastocytes and goblet cells, and performed structural and ultrastructural studies on the small intestine. We observed that 5 d of yogurt feeding was the optimal dose for improving gut barrier function and mucosal immune system in a malnutrition model. This effect was not observed with milk re-nutrition. Although the results were better for 5 d of yogurt, addition for 7 d also showed beneficial effects. Yogurt feeding in our model did not impair any gut functions. These results suggest that yogurt addition after a re-nutrition diet gives better recovery of intestinal function than the re-nutrition diet usually recommended. Although these results were obtained in an animal model, they indicate that consumption of yogurt by malnourished children might accelerate the restoration of gut function.

Animals↗
PubMed2003

The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse.

The vertebrate Cdx genes (Cdx1 Cdx2 and Cdx4 in the mouse) encode homeodomain transcription factors related to the Drosophila caudal gene. The vertebrate Cdx gene products have been implicated in the development of the posterior embryo. In particular, loss- and gain-of-function experiments suggest that Cdx members are direct regulators of Hox genes and likely impart posterior information, in part, through this mechanism. Several signaling molecules, notably retinoic acid (RA*) and members of the Wnt (wingless) and fibroblast growth factor (FGF) families, are also implicated in patterning of the posterior vertebrate embryo. Interestingly, recent work indicates that members of the Cdx family are targets of Wnt, RA and FGF signaling, suggesting that Cdx factors act to convey the activity of these signaling molecules to Hox genes. This article will briefly review Cdx expression and function, with particular emphasis on vertebrate model systems.

Animals↗
PubMedDate not supplied

Parametric display of myocardial function.

Quantitative assessment of regional heart motion has significant potential to provide more specific diagnosis of cardiac disease and cardiac malfunction than currently possible. Local heart motion may be captured from various medical imaging scanners. In this study, 3-D reconstructions of pre-infarct and post-infarct hearts were obtained from the Dynamic Spatial Reconstructor (DSR)[Ritman EL, Robb RA, Harris LD. Imaging physiological functions: experience with DSR. Philadelphia: Praeger, 1985; Robb RA, Lent AH, Gilbert BK, Chu A. The dynamic spatial reconstructor: a computed tomography system for high-speed simultaneous scanning of multiple cross sections of the heart. J Med Syst 1980;4(2):253-88; Jorgensen SM, Whitlock SV, Thomas PJ, Roessler RW, Ritman EL. The dynamic spatial reconstructor: a high speed, stop action, 3-D, digital radiographic imager of moving internal organs and blood. Proceedings of SPIE, Ultrahigh- and High-speed Photography, Videography, Photonics, and Velocimetry 1990;1346:180-91.] (DSR). Using functional parametric mapping of disturbances in regional contractility and relaxation, regional myocardial motion during a cardiac cycle is color mapped onto a deformable heart model to facilitate appreciation of the structure-to-function relationships in the myocardium, such as occurs in regional patterns of akinesis or dyskinesis associated with myocardial ischemia or infarction resulting from coronary artery occlusion.

Animals↗
PubMed2004

Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish.

Somites give rise to a number of different embryonic cell types, including the precursors of skeletal muscle populations. The lateral aspect of amniote and fish somites have been shown to give rise specifically to hypaxial muscle, including the appendicular muscle that populates fins and limbs. We have investigated the morphogenetic basis for formation of specific hypaxial muscles within the zebrafish embryo and larvae. Transplantation experiments have revealed a developmentally precocious commitment of cells derived from pectoral fin level somites to forming hypaxial and specifically appendicular muscle. The fate of transplanted somites cannot be over-ridden by local inductive signals, suggesting that somitic tissue may be fixed at an early point in their developmental history to produce appendicular muscle. We further show that this restriction in competence is mirrored at the molecular level, with the exclusive expression of the receptor tyrosine kinase met within somitic regions fated to give rise to appendicular muscle. Loss-of-function experiments reveal that Met and its ligand, hepatocyte growth factor, are required for the correct morphogenesis of the hypaxial muscles in which met is expressed. Furthermore, we demonstrate a requirement for Met signaling in the process of proneuromast deposition from the posterior lateral line primordia.

Animals↗
PubMed1999

Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate.

The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.

Acetonitriles↗
PubMed2002

The role of cell cycle-regulated expression in the localization of spatial landmark proteins in yeast.

In Saccharomyces cerevisiae, Bud8p and Bud9p are homologous plasma membrane glycoproteins that appear to mark the distal and proximal cell poles, respectively, as potential sites for budding in the bipolar pattern. Here we provide evidence that Bud8p is delivered to the presumptive bud site (and thence to the distal pole of the bud) just before bud emergence, and that Bud9p is delivered to the bud side of the mother-bud neck (and thence to the proximal pole of the daughter cell) after activation of the mitotic exit network, just before cytokinesis. Like the delivery of Bud8p, that of Bud9p is actin dependent; unlike the delivery of Bud8p, that of Bud9p is also septin dependent. Interestingly, although the transcription of BUD8 and BUD9 appears to be cell cycle regulated, the abundance of BUD8 mRNA peaks in G2/M and that of BUD9 mRNA peaks in late G1, suggesting that the translation and/or delivery to the cell surface of each protein is delayed and presumably also cell cycle regulated. The importance of time of transcription in localization is supported by promoter-swap experiments: expression of Bud8p from the BUD9 promoter leads to its localization predominantly to the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant, suggesting that some feature(s) of the Bud8p protein is also important for Bud8p function. Experiments with chimeric proteins suggest that the critical element(s) is somewhere in the extracytoplasmic domain of Bud8p.

Actins↗
PubMed2026

Reduced VEPH1 expression is associated with an invasive phenotype and poor prognosis in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) remains a clinically heterogeneous urologic malignancy, and improved biomarkers are needed to refine prognostic stratification. VEPH1 has been implicated in cancer biology, but its role in ccRCC is incompletely defined. This study aimed to investigate the expression, prognostic relevance, and functional effects of VEPH1 in ccRCC. METHODS: VEPH1 transcript expression and prognostic relevance were evaluated using The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) dataset and the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) and validated in paired ccRCC and adjacent normal renal tissues. The ability of VEPH1 transcript expression to distinguish tumor from normal tissues within the TCGA-KIRC dataset was assessed by receiver operating characteristic analysis. Gain- and loss-of-function experiments were performed in 786-O and 769-P ccRCC cells to determine the effects of VEPH1 on epithelial-mesenchymal transition (EMT)-related markers, migration, and invasion. AKT and ERK phosphorylation was evaluated by western blotting. RESULTS: VEPH1 transcript expression was significantly lower in ccRCC tissues than in normal renal tissues and distinguished tumor from normal samples within the TCGA-KIRC dataset. Low VEPH1 transcript expression was associated with poorer overall survival. Validation in 11 paired clinical specimens confirmed reduced VEPH1 messenger RNA (mRNA) and VEPH1 protein expression in tumor tissues. Functionally, VEPH1 overexpression increased E-cadherin, decreased N-cadherin, and suppressed migration and invasion, whereas partial VEPH1 knockdown produced the opposite changes. In exploratory signaling analyses, VEPH1 overexpression was associated with reduced AKT and ERK phosphorylation without altering total AKT or ERK levels. CONCLUSIONS: Reduced VEPH1 transcript expression was associated with poorer overall survival, whereas experimental VEPH1 depletion was associated with invasive and EMT-related features in ccRCC cells. VEPH1 may represent a candidate prognostic indicator in ccRCC; however, its relationship with AKT and ERK signaling and its clinical relevance require further mechanistic and independent-cohort validation.

Clear cell renal cell carcinoma (ccRCC)↗
PubMed2005

Anatomical and functional brain imaging using high-resolution echo-planar spectroscopic imaging at 1.5 Tesla.

High-resolution echo-planar spectroscopic imaging (EPSI) of water resonance (i.e. without water suppression) is proposed for anatomic and functional imaging of the human brain at 1.5 T. Water spectra with a resolution of 2.6 Hz and a bandwidth of 333 Hz were obtained in small voxels (1.7 x 1.7 x 3 mm3) across a single slice. Although water spectra appeared Lorentzian in most of the voxels in the brain, non-Lorentzian broadening of the water resonance was observed in voxels containing blood vessels. In functional experiments with a motor task, robust activation in motor cortices was observed in high-resolution T2* maps generated from the EPSI data. Shift of the water resonance frequency occurred during neuronal activation in motor cortices. The activation areas appeared to be more localized after excluding the voxels in which the lineshape of the water resonance had elevated T2* and became more non-Lorentzian during the motor task. These preliminary results suggest that high-resolution EPSI is a promising tool to study susceptibility-related effects, such as BOLD contrast, for improved anatomical and functional imaging of the brain.

Adult↗
PubMed2003

C-terminal interaction of KCNQ2 and KCNQ3 K+ channels.

Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude.

Amino Acid Sequence↗
PubMed2006

Glutamine regulates expression of key transcription factor, signal transduction, metabolic gene, and protein expression in a clonal pancreatic beta-cell line.

We have investigated the effects of prolonged exposure (24 h) to the amino acid l-glutamine, on gene and protein expression using clonal BRIN-BD11 beta-cells. Expression profiling of BRIN-BD11 cells was performed using oligonucleotide microarray analysis. Culture for 24 h with 10 mM l-glutamine compared with 1 mM resulted in substantial changes in gene expression with 148 genes upregulated more than 1.8-fold, and 18 downregulated more than 1.8-fold, including many genes involved in cellular signaling, metabolism, gene regulation, and the insulin-secretory response. Subsequent functional experiments confirmed that l-glutamine increased the activity of the Ca(2+) regulated phosphatase calcineurin and the transcription factor Pdx1. Additionally, we demonstrated that beta-cell-derived l-glutamate was released into the extracellular medium at high rates. As calcineurin is a regulator of the glutamate N-methyl-d-aspartate (NMDA) receptor activity, we investigated the action of NMDA on nutrient-induced insulin secretion, and demonstrated suppressed insulin release. These observations indicate important long-term effects of l-glutamine in regulating beta-cell gene expression, signaling, and secretory function.

Blotting, Western↗
PubMed2006

Global analysis of the transcriptional network controlling Xenopus endoderm formation.

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Animals↗
PubMed2000

Neuroanatomical organization of perceptual memory: an fMRI study of picture priming.

Neuroanatomical organization of perceptual representation in human memory system is unclear primarily because it has been studied using paradigms that have both, perceptual and conceptual components (e.g., word stem completion and word fragment completion). In the present experiment, functional magnetic resonance imaging (fMRI) technique was used to examine the pattern of cortical activation in a picture identification test in which subjects were asked to identify subliminally presented primed and novel pictures. This test is a modification of the word identification test that is considered a "pure" form of perceptual priming. Results indicate that perceptual priming is associated with reduced activation in the extrastriate cortex and that the memory for subliminally presented stimuli is processed by the same brain areas that process adequate stimuli. The activation pattern observed in picture identification test is different from that reported in the experiments of conceptual priming, suggesting that perceptual and conceptual representation of memory are supported by separate brain mechanisms.

Adult↗
PubMed1996

Glycine 699 is pivotal for the motor activity of skeletal muscle myosin.

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.

Animals↗