Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Inhibition of the ATP-dependent interaction of actin and myosin by the catalytic domain of the myosin light chain kinase of smooth muscle: possible involvement in smooth muscle relaxation

Myosin light chain kinase (MLCK) phosphorylates the light chain of smooth muscle myosin enabling its interaction with actin. This interaction initiates smooth muscle contraction. MLCK has another role that is not attributable to its phosphorylating activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a glass surface coated with phosphorylated myosin. To analyze the inhibitory effect of MLCK, the catalytic domain of MLCK was obtained with or without the regulatory sequence adjacent to the C-terminal of the domain, and the inhibitory effect of the domain was examined by the movement of actin filaments. All the domains work so as to inhibit actin filament movement whether or not the regulatory sequence is included. When the domain includes the regulatory sequence, calmodulin in the presence of calcium abolishes the inhibition. Since the phosphorylation reaction is not involved in regulating the movement by MLCK, and a catalytic fragment that shows no kinase activity also inhibits movement, the kinase activity is not related to inhibition. Higher concentrations of MLCK inhibit the binding of actin filaments to myosin-coated surfaces as well as their movement. We discuss the dual roles of the domain, the phosphorylation of myosin that allows myosin to cross-bridge with actin and a novel function that breaks cross-bridging.     

Changes in blood chemistry and hematology variables during aging in captive rhesus macaques (Macaca mulatta). J Med Primatol 30:161-173, 2001

The Primate Aging Database (PAD) is being developed to assist research using nonhuman primate models for various gerontological applications. We provide now an update of an earlier report providing data on hematological and blood chemistry values for rhesus monkeys across the adult lifespan. These data were collected from several research colonies and have been submitted to rigorous statistical analyses to identify relationships with chronological age.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.