Differences between cell lines of uterine cervical glassy cell carcinoma and large cell nonkeratinizing squamous cell carcinoma.

Cell lines were established from two uterine cervical cancers, a glassy cell carcinoma (GCC) and a large cell nonkeratinizing squamous cell carcinoma (LCSC), and studied by a variety of techniques, including histology, chromosome analysis, heterotransplantation and tumor marker analyses. There were radical differences in the morphology, heterotransplantability, production of tumor markers, etc., between the cultures of these morphologically similar cancers. The LCSC line (HKMUS) consisted of polygonal and round cells containing tonofilaments; these cells discharged tumor antigen-4 (TA-4) into the conditioned media. HKMUS was heterotransplantable into the subcutis of nude mice to form LCSC. On the other hand, the GCC line (HOKUG) consisted of round or spindle-shaped cells. HOKUG was easily transplanted into the subcutis or intraabdominal cavity of nude mice and metastasized easily. It discharged TA-4, carbohydrate antigen 125 (CA125) and neuron-specific enolase (NSE) into the conditioned media. The histologic picture of GCC revealed numerous blood vessels and a rapid proliferation of the cells. GCC, which is considered to be a mixed carcinoma having the characteristics of both squamous carcinoma and adenocarcinoma, seems to be a cancer of unpredictable prognosis as compared to LCSC, possibly due to its rapid proliferation and easy metastasis, leading to peritonitis carcinomatosa.     

Beta-actinin isoforms in various types of muscle and non-muscle tissues

We found that beta-actinin isoforms are present in various types of tissues in adult chicken by using immunoblotting after two dimensional gel electrophoresis; for this purpose, an antibody was raised against beta-actinin purified from adult chicken breast muscle (pectoralis major). One of the beta-actinin subunits, beta I, was present in all tissues we examined, i.e. skeletal (pectoralis major, semitendinosus, and anterior latissimus dorsi), cardiac, and smooth (gizzard) muscles, non-muscle (brain, liver, and kidney) tissues and blood, whereas another subunit, beta II, was present only in muscle tissues. A new subunit (designated beta III) that was found in the embryonic stages of skeletal muscle (Asami, Funatsu & Ishiwata (1988) J. Biochem. 103, 72-75) was present instead of beta II in non-muscle tissues and blood. In cardiac and smooth muscles, beta III coexisted with beta I and beta II. The antibody of beta-actinin did not cross-react to cytoplasmic beta-actinin (molecular weight, 80,000 daltons) found in kidney. It was suggested that the combination of beta I and beta III present in non-muscle tissues and blood is identical to the barbed end capping protein isolated from brain by Killiman and Isenberg (EMBO J. 1, 889-894 (1982)). It is likely that beta-actinin forms a genetic family whose constituents have an ability to cap either the pointed or barbed end of actin filaments.     

Beta-actinin: a capping protein at the pointed end of thin filaments in skeletal muscle

We examined the function of beta-actinin as a pointed end capping protein of thin filaments in skeletal muscle. An improvement in preparing beta-actinin yielded purified beta-actinin which retained its activity for more than a week. Two-dimensional gel electrophoresis showed that the two subunits, beta I and beta II, of beta-actinin are, respectively, split into two to three components (isoforms) with different isoelectric points. Polyclonal antibody was raised by injecting such purified and undenatured chicken breast muscle beta-actinin composed of several components into a rabbit. Immuno-gold labeling examination with electron microscopy of an F-actin-beta-actinin complex decorated with HMM showed that 85% of bound gold particles was on the pointed end of actin filaments, while the remaining 15% was on the barbed end. This suggests that in beta-actinin preparation pointed end and barbed end capping proteins inevitably coexist. Immunofluorescence and immunoelectron microscopy directly showed that beta-actinin is located at the pointed end of thin filaments in myofibrils; it was also suggested that a capping protein having common antigenic determinants to beta-actinin is located at Z-line. Thus, the physiological function of beta-actinin as a pointed end capping protein was examined as follows: When beta-actinin was dissociated from the pointed end of thin filaments in an I-Z-I brush by using a high salt solution, thin filaments could be disassembled at the pointed ends at concentrations of exogenous actin lower than a critical value. At a physiological ionic strength, these salt-washed thin filaments gradually shortened at a constant rate of about 45 nm/h. Both the association and dissociation of monomeric actin at the pointed end were suppressed by the rebinding of exogenous beta-actinin. The main physiological role of beta-actinin is therefore to stabilize thin filaments in the sarcomere by preventing addition and removal of actin monomers at the pointed filament end.     

Key residues on microtubule responsible for activation of kinesin ATPase

Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged‐to‐alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11–12 (H11–12) loop and H12 of α‐tubulin, and the negatively charged residues in H12 of β‐tubulin. Mutation in the α‐tubulin‐binding site results in a deceleration of ATP hydrolysis (k_cat), whereas mutation in the β‐tubulin‐binding site lowers the affinity for MTs (K_0.5MT). The residue E415 in α‐tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing α‐E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.     

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca²⁺ and incubated overnight at 4 °C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca²⁺-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.     

Protein kinase A–dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during β-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca²⁺ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca²⁺ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca²⁺ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca²⁺ sensitivity, but also abolished the Ca²⁺-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca²⁺ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca²⁺ sensitivity, and PKA further decreased Ca²⁺ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca²⁺ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.     

Synthesis and TNF-alpha inducing activities of mycoloyl-arabinan motif of mycobacterial cell wall components

The extract of the cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) from Mycobacterium bovis is known to be an activator of innate immunity. Synthesis of pentaarabinofuranoside as part of the arabinan moiety of BCG-CWS was achieved by double alpha-arabinofuranosylation followed by double beta-arabinofuranosylation with orthogonally protected donors. Mycolic esters of the arabinan in the terminal lipo-arabinan motif of BCG-CWS were synthesized through alkylation of unprotected mycolic acid with bis- and tetra-tosylates of pentaarabinofuranoside. A series of compounds were subjected to a tumor necrosis factor alpha (TNF-alpha) secretion-inducing assay, disclosing aspects of the structure-activity relationship which should be useful in finding the site of the activity.     

Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation

Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament “on–off” switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca²⁺ sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca²⁺ sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca²⁺-dependent on–off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on–off switching, but not the Ca²⁺-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.