Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.     

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.     

G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.     

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of (R)-glyceraldehyde 3-phosphate in D2O

The product distributions for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D(2)O at pD 7.5-7.9 catalyzed by triosephosphate isomerase (TIM) from chicken and rabbit muscle were determined by (1)H NMR spectroscopy. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen (49% of the enzymatic products), [1(R)-(2)H]-DHAP from isomerization with incorporation of deuterium from D(2)O into C-1 of DHAP (31% of the enzymatic products), and [2(R)-(2)H]-GAP from incorporation of deuterium from D(2)O into C-2 of GAP (21% of the enzymatic products). The similar yields of [1(R)-(2)H]-DHAP and [2(R)-(2)H]-GAP from partitioning of the enzyme-bound enediol(ate) intermediate between hydron transfer to C-1 and C-2 is consistent with earlier results, which showed that there are similar barriers for conversion of this intermediate to the alpha-hydroxy ketone and aldehyde products (Knowles, J. R., and Albery, W. J. (1977) Acc. Chem. Res. 10, 105-111). However, the observation that the TIM-catalyzed isomerization of GAP in D(2)O proceeds with 49% intramolecular transfer of the (1)H label from substrate to product DHAP stands in sharp contrast with the 相似文献   

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4)

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.     

The P450cam G248E mutant covalently binds its prosthetic heme group

Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme. To determine whether there are additional requirements for covalent bond formation in the P450 enzymes, a glutamic acid or an aspartic acid has been introduced into P450(cam) close to the heme 5-methyl group. Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom, as reported for a comparable P450(BM3) mutant [Girvan, H. M., Marshall, K. R., Lawson, R. J., Leys, D., Joyce, M. G., Clarkson, J., Smith, W. E., Cheesman, M. R., and Munro, A. W. (2004) J. Biol. Chem. 279, 23274-23286]. The two P450(cam) mutants have low catalytic activity, but in contrast to the P450(BM3) mutant, incubation of the G248E (but not G248D) mutant with camphor, putidaredoxin, putidaredoxin reductase, and NADH results in partial covalent binding of the heme to the protein. No covalent attachment is observed in the absence of camphor or any of the other reaction components. Pronase digestion of the G248E P450(cam) mutant after covalent attachment of the heme releases 5-hydroxyheme, establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling. The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes, but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.     

Expression of multiple Src family kinases in sea urchin eggs and their function in Ca release at fertilization

Egg activation at fertilization in deuterostomes requires a rise in intracellular Ca²⁺, which is released from the egg's endoplasmic reticulum. In sea urchins, a Src Family Kinase (SpSFK1) is necessary for the PLCγ-mediated signaling event that initiates this Ca²⁺ release (Giusti, A.F., O'Neill, F.J., Yamasu, K., Foltz, K.R. and Jaffe, L.A., 2003. Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization. Dev. Biol. 256, 367-378.). Annotation of the Strongylocentrotus purpuratus genome sequence led to the identification of additional, predicted SFKs (Bradham, C.A., Foltz, D.R., Beane, W.S., Amone, M.I., Rizzo, F., Coffman, J.A., Mushegian, A., Goel, M., Morales, J., Geneviere, A.M., Lapraz, F., Robertson, A.J., Kelkar, H., Loza-Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lepage, T., Gache, C., McClay, D.R., Manning, G., 2006. The sea urchin kinome: a first look. Dev. Biol. 300, 180-193.; Roux, M.M., Townley, I.K., Raisch, M., Reade, A., Bradham, C., Humphreys, G., Gunaratne, H.J., Killian, C.E., Moy, G., Su, Y.H., Ettensohn, C.A., Wilt, F., Vacquier, V.D., Burke, R.D., Wessel, G. and Foltz, K.R., 2006. A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation. Dev. Biol. 300, 416-433.). Here, we describe the cloning and characterization of these 4 additional SFKs and test their function during the initial Ca²⁺ release at fertilization using the dominant-interfering microinjection method coupled with Ca²⁺ recording. While two of the new SFKs (SpFrk and SpSFK3) are necessary for Ca²⁺ release, SpSFK5 appears dispensable for early egg to embryo transition events. Interestingly, SpSFK7 may be involved in preventing precocious release of Ca²⁺. Binding studies indicate that only SpSFK1 is capable of direct interaction with PLCγ. Immunolocalization studies suggest that one or more SpSFK and PLCγ are localized to the egg cortex and at the site of sperm-egg interaction. Collectively, these data indicate that more than one SFK is involved in the Ca²⁺ release pathway at fertilization.     

Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.     

A roof plate-dependent enhancer controls the expression of Homeodomain only protein in the developing cerebral cortex

The smallest known homeodomain protein, Homeodomain only protein (Hop), was identified and described here as a temporally and spatially restricted gene in the neurogenic regions of the developing murine CNS including the cerebral cortex. Furthermore, an evolutionarily conserved 418 base pair upstream cis-regulatory DNA sequence was found to confine the Hop expression to the CNS of transgenic mice, but not to the heart which is the second major Hop expressing organ Chen, F., Kook, H., Milewski, R., Gitler, A.D., Lu, M.M., Li, J., Nazarian, R., Schnepp, R., Jen, K., Biben, C., Runke, G., Mackay, J.P., Novotny, J., Schwartz, R.J., Harvey, R.P., Mullins, M.C., Epstein, J.A., 2002. Hop is an unusual homeobox gene that modulates cardiac development. Cell 110, 713-723; Shin, C.H., Liu, Z.P., Passier, R., Zhang, C.L., Wang, D.Z., Harris, T.M., Yamagishi, H., Richardson, J.A., Childs, G., Olson, E.N., 2002. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110, 725-735. The forebrain enhancer activity was successfully reproduced in vitro utilizing a combination of the electroporation and the organotypic brain culture method. Using this approach, the minimal requirement for the forebrain-specific enhancer sequence was delineated down to 200 base pairs. We further demonstrate that the Hop enhancer activity is inducible ectopically in a transgenic tissue by wild-type roof plate transplantation in vitro. Thus Hop is regulated in the forebrain by a so far unidentified paracrine signaling factor from the roof plate. Furthermore, the identified enhancer sequence provides an important tool for the targeted expression of transgenes in the medial cortex and the cortical hem.     

A 1H-NMR study of electronic structure of the active site of Galeorhinus japonicus metmyoglobin

The ferric high-spin form of the myoglobin from the shark Galeorhinus japonicus, which possesses a Gln residue at the distal site instead of the usual His residue, has been studied by 1H-NMR spectroscopy. Using the heme meso-proton (C5H, C10H, C15H and C20H) resonance shift as a diagnostic probe for identifying the coordination system of the iron center in ferric high-spin form of hemoprotein, it has been shown that G. japonicus metmyoglobin (metMb) possesses the pentacoordinated active site. The pH-dependence study of NMR spectra of G. japonicus metMb revealed the appearance of the hydroxyl form of metMb at high pH, indicating that the protein undergoes the transition between the acidic and alkaline forms. The pK value and the rate for this acid-alkaline transition in G. japonicus metMb were found to be approximately 10 and much less than 4 x 10(2) s-1, respectively. Since the pK value of the acid-alkaline transition for the pentacoordinated heme in Aplysia limacina metMb is 7.8 [Giacometti, G.M., Das Ros, A., Antonini, E. & Brunori, M. (1975) Biochemistry 14, 1584-1588] and that of the hexacoordinated heme in sperm whale metMb is 9.1 [Brunori, M., Antonini, E., Fasella, P., Wyman, J. & Rossi-Fanelli, A. (1968) J. Mol. Biol. 34, 497-504], the OH- affinity of the ferric heme iron does not appear to depend on its coordination system. The acid-alkaline transition rate in A. limacina metMb was reported to be much less than 1.5 x 10(2) s-1 [Pande, U., La Mar, G.N., Lecomte, J.T.J., Ascoli, F., Brunori, M., Smith, K.M., Pandey, R.K., Parish, D.W. & Thanabal, V. (1986) Biochemistry 25, 5638-5646] and therefore a slow transition rate may be unique to the pentacoordinated active site of Mb.     

Characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398-23401.) Here we show that the selectivity for the binding of different NH(2)-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl > fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF > fMLF-O-methyl approximately fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR (93)VRK(95) (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 10428-10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group of fMLF ion-paired with Arg-205.