Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.     

Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1

Activated Cdc42-associated kinase-2 (ACK-2) is a non-receptor tyrosine kinase that appears to be a highly specific target for the Rho-related GTP-binding protein Cdc42. In order to understand better how ACK-2 activity is regulated in cells, we have expressed epitope-tagged forms of this tyrosine kinase in COS-7 and NIH3T3 cells. We find that ACK-2 can be activated by cell adhesion in a Cdc42-dependent manner. However, unlike the focal adhesion kinase, which also is activated by cell adhesion, the activation of ACK-2 is F-actin-independent and does not require cell spreading. In addition, overexpression of ACK-2 in COS-7 cells did not result in the stimulation of extracellular signal-regulated kinase activity but rather activated the c-Jun kinase. Both anti-integrin beta1 antibody and RGD peptides inhibited the activation of ACK-2 by cell adhesion. In addition, ACK-2 was co-immunoprecipitated with integrin beta1. Overall, these findings suggest that ACK-2 interacts with integrin complexes and mediates cell adhesion signals in a Cdc42-dependent manner.     

Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1

Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.     

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.     

Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) and is known as the "10-23" general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3'-3'-internucleotide inversion at the 3'-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.     

Preferential expression of biotransformation enzymes in the olfactory organs of Drosophila melanogaster, the antennae

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that in Drosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe five Drosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophila families suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates.     

A human genome map of comparative anchor tagged sequences.

Effective comparative mapping inference utilizing developing gene maps of animal species requires the inclusion of anchored reference loci that are homologous to genes mapped in the more "gene-dense" mouse and human maps. Nominated anchor loci, termed comparative anchor tagged sequences (CATS), have been ordered in the mouse linkage map, but due to the dearth of common polymorphisms among human coding genes have not been well represented in human linkage maps. We present here an ordered framework map of 314 comparative anchor markers in humans based on mapping analysis in the Genebridge 4 panel of radiation hybrid cell lines, plus empirically optimized CATS PCR primers which detect these markers. The ordering of these homologous gene markers in human and mouse maps provides a framework for comparative gene mapping of representative mammalian species.     

Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum

We analyzed the dynamics of splenic T-lymphocyte function in relation to hepatopathologic changes in C3H/Hc mice, experimentally infected with Schistosoma japonicum. Vigorous granuloma formation was observed at 7 wk postinfection. At 10 wk postinfection, granuloma formation entered into the down-modulation stage, as represented by the diminished granuloma size. The Th2 response was activated when eggs appeared in the liver, whereas Th1 responses were depressed and the proliferation of T lymphocytes was decreased. The level of IgG antibodies to the worm and egg antigens rose continually after infection. Interleukin-12 treatment of infected mice inhibited Th2 responses and T-cell proliferation, decreased granuloma formation and fibrosis, but had no effect on the fecundity of the worms. These data suggest that egg deposition is the major factor driving Th2 responses, depressing Th1 cytokine expression as well as T-cell proliferation in S. japonicum-infected mice.     

Testing for age-at-onset anticipation with affected parent-child pairs

The tendency for the onset of a genetic disease to occur at progressively earlier ages or with progressively greater severity in successive generations is known as anticipation. Following the discovery of trinucleotide repeat expansion as a plausible genetic mechanism for anticipation, interest in testing for anticipation has increased. Studies of anticipation can be biased when parents with late onset or children with early onset are preferentially ascertained. This paper presents a nonparametric approach to testing for age-at-onset anticipation that adjusts for such preferential ascertainment. The approach is illustrated through application to data on panic disorder.