Methylisocyanate and actin polymerization: the in vitro effects of carbamylation.

Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4 degrees C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.

Homology among beta-crystallins: monoclonal antibodies to beta-heavy crystallin.

The mouse monoclonal antibodies, prepared with rat beta H-crystallin as parent antigen, show cross-reactivity to specific subunits in beta H-, beta L1-, and beta L2-crystallin but no reactivity to either alpha- or gamma-crystallins. Antigenic homology among the beta-crystallin subunits was demonstrated by comparison of the topographic distributions of peptides on two-dimensional electrophoretograms subjected to either staining or an ELISA of the immunoblots.

Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue.

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.

Na+-K+-ATPase in rat vascular smooth muscle cell grown in vitro.

This study has focused on the characteristics of the Na+-K+-ATPase in in vitro preparations of vascular smooth muscle cells (VSMCs) derived from the rat carotid artery. The maximum velocity of enzyme reaction (Vmax) for the specific activity of the enzyme in the VSMCs' preparations was 2.36 +/- 0.04 (SE) mumol Pi X mg cell protein-1 X h-1 or 0.82 +/- 0.02 mumol Pi X 10(6) cells-1 X h-1. The activation of the enzyme by potassium, sodium and ATP has been investigated. The half-maximal values for potassium and sodium activation of the enzyme in the preparations were 1.18 and 10-20 meq/l, respectively. The respective Vmax values for potassium and sodium activation were reached at concentrations of 4-10 and 80-100 meq/l. The Michaelis constant for ATP was 0.83 mM. Calcium exerted a potent inhibition on the activity of the enzyme (I50 at 1 mM). It has been concluded that the Na+-K+-ATPase kinetic pattern in in vitro preparations of VSMCs is quite similar to that observed in homogenates or subcellular fractions of other tissues.

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.

Analysis of the mechanism of fast axonal transport by intracellular injection of potentially inhibitory macromolecules: evidence for a possible role of actin filaments.

Although actin is thought to participate in several types of cell motility other than muscle contraction, no direct evidence has linked it to the force-generating mechanism for fast axonal transport. We have obtained evidence for the involvement of actin by microinjecting, into the serotonergic giant cerebral neuron of Aplysia, two preparations that have been shown to depolymerize actin filaments. One is a fraction of rabbit serum containing a heat-labile gamma globulin that affects actin polymerization in a manner similar to that of cytochalasin and several proteins that are thought to regulate the length of actin filaments. The other is bovine pancreatic DNase I which binds to actin stoichiometrically. Both preparations substantially decreased the transport of storage vesicles containing [3H]serotonin. Phalloidin, a toxic fungal peptide that binds to actin filaments but stabilizes rather than depolymerizes them, did not inhibit transport. We have not yet determined whether the inhibition od transport occurs during export of [3H]serotonin from the cell body into the axon or during translocation along the axon. Nevertheless, these observations provide a promising experimental indication that actin is involved in fast axonal transport.

An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins.

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica. We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin. Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form.

Peroxidase technique for the detection of photochemical lesions in intracellular deoxyribonucleic acid.

The widespread use of phototherapy for the prevention and treatment of neonatal hyperbilirubinemia has generated some conxern as physiologic substances other than bilirubin may be photoactivated. Little information is available on the long term toxicity of these photodecomposition products. Recent observations of the in vitro DNA-modifying activity of phototherapy lights has encouraged us to develop laboratory procedures which can identify and quantitate these light-induced alterations. The purpose of the present study was to develop a technique capable of detecting photochemical changes in the genetic material of human cells in tissue culture. The results demonstrate that the antinucleoside peroxidase staining procedure is capable of detecting changes in the DNA of human cells exposed to physiologic (riboflavin) and nonphysiologic (methylene blue) photosensitizing agents in the presence of light with a fluence rate (450 nm) of 141 muW-cm2.

Localization of 5-methylcytosine in human metaphase chromosomes by immunoelectron microscopy.

Human metaphase chromosomes, fixed in methanol-acetic acid, were ultraviolet irradiated to produce single-stranded regions of chromosomal DNA and treated with anti-5-methylcytidine. Using an immunoperoxidase procedure, regions of antibody binding were readily visualized by light microscopy in the centromeric heterochromatin regions of chromosomes 1, 9, 16, the short arm of chromosome 15, and in the distal portion of the Y. Electron microscopic visualization of the same whole mount chromosome preparations transferred to formvarcoated grids revealed additional details of the distribution and arrangement of 5-methylcytosine. A helical arrangement of 5-methylcytosine residues was seen below the centromere of chromosome 1. The Y chromosome showed a concentration of 5-methylcytosine residues on the distal long arm, and in areas just below and slightly above the centromere. In all the above chromosomes, especially chromosome 15, additional 5-methylcytosine residues were detected as isolated foci along the arms. Our findings support the concept that clusters of similar purine or pyrimidine residues exist along the arms of condensed metaphase chromosomes, with the possibility that concentrations of 5-methylcytosine residues might have been enhanced at the surface of the chromosomes during the condensation process.