Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5''-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

ADP-ribosylation of nuclear proteins in normal lymphocytes and in low-grade malignant non-Hodgkin lymphoma cells

Normal lymphocytes and lymphocytes from patients with low-grade malignant non-Hodgkin lymphoma were isolated from blood by a Percoll gradient procedure. Absence of cell proliferation in both cell types was indicated by very low [3H]thymidine incorporation rates. Determination of endogenous protein-bound single ADP-ribose residues by a radioimmunoassay revealed that the leukemic cells had 2.5-times lower levels of the NH2OH-sensitive and a 4-fold lower amount of NH2OH-resistant ADP-ribose . protein conjugate subfractions, respectively, than normal lymphocytes. By contrast, "total" ADP-ribose transferase activity, as measured in homogenates or permeabilized cells in the presence of DNase, was two-times higher in leukemic cells, whereas activity determined in permeabilized cells in the absence of added DNase was practically identical in both cell types. The apparent discrepancy between ADP-ribose transferase activity and endogenous levels of protein-bound single ADP-ribose residues may be explained in part by an enzyme inhibitor present in normal human lymphocytes. NAD + NADH levels were decreased 2.5-fold in the leukemic cells. This decrease, however, does not explain the reduced levels of mono(ADP-ribose) . protein conjugates since the ratio of protein-bound single ADP-ribose residues to NAD is distinctly different in leukemic lymphocytes compared to normal lymphocytes.     

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Determination of the net exchange rate of tubulin dimer in steady-state microtubules by fluorescence correlation spectroscopy

The microtubule cytoskeleton plays an important role in eukaryotic cells, e. g., in cell movement or morphogenesis. Microtubules, formed by assembly of tubulin dimers, are dynamic polymers changing randomly between periods of growing and shortening, a property known as dynamic instability. Another process characterizing the dynamic behaviour is the so-called treadmilling due to different binding constants of tubulin at both microtubule ends. In this study, we used tetramethylrhodamine (TMR)-labeled tubulin added to microtubule suspensions to determine the net exchange rate (NER) of tubulin dimers by fluorescence correlation spectroscopy (FCS) as a measure for microtubule dynamics. This approach, which seems to be suitable as a screening system to detect compounds influencing the NER of tubulin dimers into microtubules at steady-state, showed that taxol, nocodazole, colchicine, and vinblastine affect microtubule dynamics at concentrations as low as 10(-9)-10(-10) M.     

Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.     

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post‐natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.