Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture

A model of the structure of vertebrate Z-lines and Z-line analogs is introduced and supported by evidence from electron microscope studies of wide Z-lines (rat and feline soleus, and feline and canine cardiac muscles), narrow Z-lines (guppy, newt and frog skeletal muscles), and Z-rods (from a patient with nemaline myopathy and from cardiac muscles of aged dog). The model is based on a pair of Z-filaments (termed a Z-unit), which are linked near their centers at a 90 degrees angle and form bridges between neighboring antipolar thin (actin) filaments. A square lattice of four Z-filament pairs (the basic structure of the Z-line, termed a Z-line unit) defines the geometrical position of the I-square unit. In this native state of the Z-line, small square and large square net forms appear in cross-section. Other cross-sectional patterns of Z-lines, including basket-weave and diagonal-square net patterns, can be explained by detachment of the Z-filament from the Z-filament binding region within each Z-filament pair due to chemical or physical stress. Dissection of Z-lines and Z-line analogs with calcium-activated neutral protease provides evidence that the width of all wide Z-line structures is determined by the amount of overlap of antipolar thin filaments from adjacent sarcomeres. Longitudinal patterns of narrow and wide Z-lines are shown and described in relation to the model. To test the proposed model, the dynamics of the Z-line unit structure were computer-simulated. An attempt was made to correlate longitudinal (z direction) and cross-sectional (x and y directions) patterns and to determine the amount of movement of thin or Z-filaments that is required to explain the diversity observed in cross-sectional patterns of Z-lines. The computer simulations demonstrated that the structural transitions among the small square, and therefore large square net, as well as basket-weave and diagonal-square net forms seen in cross-sections could be caused by movements of thin filaments less than 10 nm in any direction (x, y or z).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fimbriae from the oral anaerobe Bacteroides gingivalis: physical, chemical, and immunological properties.

Circular dichroism spectra indicated the predominance of beta-sheet structure in Bacteroides gingivalis fimbriae regardless of the presence of sodium dodecyl sulfate. By using a computer program, the alpha-helix, beta-sheet, and beta-turn contents and the remainder were estimated to be 0, 55, 18, and 27%, respectively, judging from the circular dichroism spectra of the fimbriae. Heating for 5 min at 100 degrees C in sodium dodecyl sulfate was necessary to denature the fimbriae into their constituent protein (fimbrilin) monomers with a reduced content of beta-sheet structure. The amino-terminal amino acid sequence of the fimbrilin was different from partial or complete amino acid sequences of fimbrilins so far determined from Bacteroides nodosus, which falls into the same nonfermentative species of the genus Bacteroides as B. gingivalis, and from various other bacteria. Fimbrilin monomers had an isoelectric point of 6.0. Examination of antibodies against fimbriae and sodium dodecyl sulfate-denatured fimbrilin by enzyme-linked immunosorbent assay reinforced a previous notion (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) that different sets of antigenic determinants seemed to be exposed on their surfaces.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Phenazine methosulfate stimulation of ouabain-sensitive Rb+ uptake by HeLa cells: effects of respiratory inhibitors, anaerobiosis, and ascorbate

phenazine methosulfate (PMS) stimulates ouabain-sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 microM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS-stimulated ouabain-sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na+K-pump activity is closely related to the cellular redox state.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.     

Purification and characterization of two kinds of porins from the Enterobacter cloacae outer membrane.

Two major outer membrane proteins of Enterobacter cloacae 206 were purified and identified as porins by using reconstituted vesicles. The 37-kilodalton porin forms a channel with a radius of 0.6 nm, which prefers positively charged substances to negatively charged ones, whereas the 39- to 40-kilodalton porin forms a larger channel with a radius of 0.8 nm, which has weaker selectivity for electric charges.     

Isolation and characterization of human placenta fibronectin

Fibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein.     

Oxidation of nitroxide radicals by the reaction of hemoglobin with hydrogen peroxide

The ESR signal of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine in hemoglobin solution decreased drastically by the addition of hydrogen peroxide. The results of ion-exchange chromatography and sodium tetraphenylborate on the reaction solution showed an oxidation of the nitroxide radical to cation form. On the basis of the comparison of thin layer-chromatogram with the reaction products of the nitroxide radicals with HCl or Br2, the formation of 4-hydroxy-1-oxo-2,2,6,6- tetramethylpiperidinium cation was demonstrated. This result was supported by the 13C NMR measurement.     

Genetic analysis of three additional fla genes in Salmonella typhimurium

In Salmonella typhimurium, 27 fla genes responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, to fla region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS, flbC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.