Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.     

Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.     

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.     

Reconstruction of a type-B configuration monkey Sertoli cell: size, shape, and configurational and specialized cell-to-cell relationships

A model of a stage-V monkey Sertoli cell was reconstructed from electron micrographs taken of semiserial sections. The configuration (type-B) was one in which spermatids were positioned near the lumen, their heads occupying shallow cylindrical recesses at the apical portion of the Sertoli cell. The cell volume was calculated to be 4,100 micron3, the surface area 2,400.68 micron2, and the surface-to-volume ratio 0.58:1. The reconstructed cell extended from the basal lamina to the tubular lumen and was generally of the tall columnar type although its surface contour was highly irregular. The dimensions of the cell [centripetal (68.46 micron), circumferential (18.40 micron), and longitudinal (21.63 micron)] were determined and cell surfaces designated. Relative and absolute surface areas of the reconstructed cell which faced other Sertoli cells, germ cells, basal lamina, and tubular lumen were calculated. Junctions and surface specializations were enumerated, catalogued, and depicted on diagrams of the cell surface. Where appropriate, type-A rat and type-B monkey Sertoli cells were compared and discussed. Morphometry was utilized to analyze the relative surface areas of germ cells adjoining the reconstructed cell to determine the percentage of their surface facing cellular and acellular elements, and these data were compared to data obtained for the rat.     

Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded alpha-chymotrypsin

Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease alpha-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be due to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Evaluation of the control of Myobia musculi infestations on laboratory mice with permethrin

The efficacy of permethrin against Myobia musculi (Schrank) (Acari:Myobiidae) infestations on mice was evaluated using four different dose delivery methods. In all methods, an attempt was made to deliver 0.5 mg of active permethrin weekly to each mouse. Successful control was achieved with topical or environmental treatment with 0.25% permethrin dust and by dipping the mice into a 0.6 g/l permethrin emulsion. The use of an emulsion applied to the bedding produced the least satisfactory control. Untreated mice had an average infestation rate of 57.3% throughout the study. No significant differences were seen between treated and untreated groups in either body weight or histopathology of the liver, lung, or kidney.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

2'-Nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2'-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2'-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2'NDG, the potent activity of 2'-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2'-nor-cGMP produced small amounts of 2'NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.