Chemomechanical transduction in the actomyosin molecular motor by 2',3'-dideoxydidehydro-ATP and characterization of its interaction with myosin subfragment 1 in the presence and absence of actin.

The effect of torsional freedom about the N-glycoside bond of ATP in the ability of the nucleoside triphosphate to support chemomechanical transduction (Takenaka et al., 1978) has been investigated by examining the ability of the nucleotide analogue 2',3'-dideoxy-2',3'-didehydro-ATP (1b, enf-ATP) to act as a substrate for myosin subfragment 1 in the presence and absence of actin and to support actin sliding in the standard in vitro motility assay. By converting the ribosyl ring of the natural substrate to the rigid and almost planar enofuranosyl ring, effects on torsional freedom about the N-glycoside bond due to changes in ribosyl ring pucker and/or by steric interferences of the protons attached to the 2' and 3' carbons are eliminated allowing for increased torsional freedom about the N-glycoside bond.

Identification of myosin III as a protein kinase.

Drosophila ninaC gene encodes myosin homologous proteins which are classified as myosin III of the myosin superfamily, yet the physiological and biochemical function of myosin III has not characterized. We report here that myosin III does exhibit protein kinase activity. The kinase homologous domain (MYOIIIPK) of myosin III was expressed in the baculovirus expression system and purified to homogeneity.

Enhancement of smooth muscle contraction with protein phosphatase inhibitor 1: activation of inhibitor 1 by cGMP-dependent protein kinase.

New method for purification of phosphatase inhibitor 1 (PPI-1) was developed which avoid the phosphorylation of PPI-1 during the purification and provides a high yield of highly pure preparation. Using this preparation, it was shown that PPI-1 was stoichiometrically phosphorylated by cGMP-dependent protein kinase at Thr-35 and the phosphorylated PPI-1 potently inhibited protein phosphatase 1. Addition of the phosphorylated PPI-1 to beta-escin-skinned single smooth muscle cells resulted in force development of the cells at the submaximal pCa2+.

Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor gamma chain.

The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region.

Functional expression of mammalian myosin I beta: analysis of its motor activity.

The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M.

Developmental and tissue distribution of expression of nonmuscle and smooth muscle isoforms of myosin light chain kinase.

Using a combination of Northern and Western blotting and RT-PCR, we demonstrate the existence of a high molecular mass MLCK, which is expressed during chicken embryogenesis. It is expressed in developing smooth muscle containing tissues, and is detected at low concentrations in adult tissues. Direct sequencing of the RT-PCR product from embryonic tissue RNA revealed that the embryonic, high molecular mass MLCK is indeed the previously cloned "nonmuscle MLCK".

Structural basis for actomyosin chemomechanical transduction by non-nucleoside triphosphate analogues.

Methylation of 2-[(2,4-dinitrophenyl)amino]ethyl triphosphate (dNOTP) was found to abolish its ability to support actin sliding in the in vitro motility assay. A comparative study of the interaction of myosin subfragment 1 (S1) and actoS1 with methylated (MdNOTP) and non-methylated dNOTP was undertaken. Both analogues were shown to be substrates for S1 NTPase in the presence of K+/EDTA, Ca2+, or Mg2+, although their rates of hydrolysis in the presence of the divalent cations were significantly greater than that occurring for ATP.

Characterization of Ca2+/calmodulin-dependent protein kinase II from smooth muscle.

We have characterized chicken gizzard smooth muscle Ca2+/calmodulin-dependent protein kinase II (CaM-PKII) with particular focus on its autophosphorylation. The autophosphorylation of smooth muscle CaMPKII produced a partially constitutively active enzyme, as occurs with the alpha- and beta-isoforms of this enzyme, but the autophosphorylation kinetics were significantly slower. Phosphorylation during the initial rapid phase coincided with the production of constitutively active enzyme.

Pseudosubstrate sequence may not be critical for autoinhibition of smooth muscle myosin light chain kinase.

It has been hypothesized that basic residues in the autoinhibitory region of myosin light chain (MLC) kinase, which resemble the substrate sequence, interact with the catalytic core via charge interaction and thus inhibit the kinase activity (pseudosubstrate inhibitory hypothesis). In the present study, we produced seven MLC kinase mutants in which the residues in the autoinhibitory region are deleted to various extents, and determined the residues crucial for the autoinhibition of the kinase activity. The activities of MT799 (1-799) and MT796 (1-796) were completely inhibited, whereas MT793 (1-793), MT791 (1-791), MT787 (1-787) and MT783 (1-783) were constitutively active.

Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament.

The structure of smooth muscle thin filament was examined by various electron microscopy techniques, with special attention to the mode of caldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in reconstituted thin filament was observed as fine filamentous projections from thin filament.

Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells.

Smooth muscle hypertrophy is often found in tissue subjected to abnormal physical stress. To determine if physical stress (strain) per se could increase the contractile potential of airway smooth muscle (ASM), we compared cultured ASM cells subjected to strain to control cells (no strain) for rates of 1) myosin light chain kinase (MLCK)-mediated myosin light chain (LC20) phosphorylation, 2) actin-activated myosin ATPase, and 3) myosin light chain phosphatase-mediated myosin dephosphorylation. Lysates from strained cells showed increases in both LC20 phosphorylation activity and actomyosin ATPase activity but decreased rates of phosphatase-dependent myosin dephosphorylation.

Influence of an anti-angiogenic treatment on 9L gliosarcoma: oxygenation and response to cytotoxic therapy.

Tissue oxygen tensions were measured in subcutaneously growing rat 9L gliosarcoma under normal air and carbogen breathing conditions prior to and after i.v. administration of a perflubron emulsion.

Requirement of the two-headed structure for the phosphorylation dependent regulation of smooth muscle myosin.

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect cells using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln945-Asp991 are critical for the formation of the two-headed structure.

Autophosphorylation of smooth muscle myosin light chain kinase at its regulatory domain.

Autophosphorylation of smooth muscle myosin light chain kinase was initially reported by Foyt et al. [Foyt, H. L.

Inhibition of 20-kDa myosin light chain exchange by monoclonal antibodies against 17-kDa myosin light chain.

Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies.

Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by x-ray solution scattering.

We have shown for the first time that the myosin head (subfragment-1, S1), the energy-transducing component in the actomyosin motor system undergoes a distinct shape change during hydrolysis of ATP using x-ray solution scattering techniques. Among various analogs for intermediate states of the S1 ATPase cycle, the complexes with MgADP and vanadate (S1.ADP.

Effect of caldesmon on the assembly of smooth muscle myosin.

Smooth muscle myosin filaments are much less stable than the skeletal muscle counterpart. Smooth myosin requires higher concentration of Mg2+ than skeletal myosin to form thick filaments and addition of ATP disassembles the dephosphorylated smooth muscle myosin filaments into monomers but not phosphorylated ones. We found that the addition of caldesmon to dephosphorylated myosin induced the formation of the filaments under the conditions where myosin by itself is soluble or disassembled.

Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearing EMT-6 mammary carcinoma.

The ability of the antiangiogenic agents TNP-470 and minocycline, singly or in combination, to potentiate the antitumor effects of several cytotoxic therapies was assessed in the murine EMT-6 mammary carcinoma as well as in two drug resistant sublines of that tumor designated EMT-6/CTX and EMT-6/CDDP. The antiangiogenic agents alone or in combination did not alter the growth of the tumors. However, their administration along with cyclophosphamide, CDDP, or thiotepa substantially increased the tumor growth delay produced by these cytotoxic therapies in tumors responsive to the drugs--the increase was about 2-fold for TNP-470 and minocycline together.

Transient regression in lymphocyte count in B-cell chronic lymphocytic leukemia after viral infection.

Transient regression in the lymphocyte count of a patient with B-cell chronic lymphocytic leukemia (B-CLL) after viral infection is reported. A similar event occurred under natural interferon-alpha (IFN-alpha) treatment. It was confirmed that the event was not caused by a direct cytotoxic effect of IFN-alpha by analyzing the DNA fragmentation to estimate apoptotic and necrotic cell death before and after the administration of IFN-alpha.

Increased efficacy of chemo- and radio-therapy by a hemoglobin solution in the 9L gliosarcoma.

Tissue oxygen tensions were measured in the rat 9L gliosarcoma under conditions of normal air breathing or carbogen breathing and after intravenous administration of a hemoglobin solution with air breathing or carbogen breathing. Administration of the hemoglobin decreased the level of hypoxia in the tumors. Treatment of the animals with the antiangiogenic combination of TNP-470 and minocycline also increased tumor oxygenation compared with untreated controls.

Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle.

The permeability to high molecular weight (IgG, 150 kD) proteins of the plasma membrane of receptor-coupled smooth muscles permeabilized with beta-escin was determined using confocal microscopy of immunofluorescent tracers and measurement of lactate dehydrogenase (LDH, 135-140 kD) leakage. Permeabilized strips of rabbit portal vein and guinea pig ileum were incubated in a relaxing solution containing mouse anti-smooth muscle alpha-actin antibody and immunostained with F(ab')2 labeled with tetramethyl rhodamine isothiocyanate. Confocal light microscopy of Triton X-100 and beta-escin permeabilized cells showed homogeneous staining of the cytoplasm, whereas in alpha-toxin treated and intact preparations only damaged cells at the edges of the strips were stained.

Pre-operative estimation of complete resection for patients with oesophageal carcinoma.

Three hundred and seventy-nine patients were studied retrospectively regarding the possibility of a complete resection of the oesophageal carcinoma based on the combined findings of pre-operative oesophagogoraphy and computed tomography (CT). One hundred and four out of 129 patients (96.1%) having lesions which did not demonstrate all three of the aforementioned factors (a lesion shorter than 8 cm, a normal oesophageal axis, and normal contact of the lesion with neighboring organs in CT) underwent a complete resection of the oesophageal lesion.

Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin.

The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation.