Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase

In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg²⁺ with Mn²⁺ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn²⁺ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or δ polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3′) guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3′ guanine of the lesion. Furthermore, substitution of MgCl₂ with MnCl₂ greatly inhibited the 3′ to 5′ exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.     

Responsiveness of mesolimbic, mesocortical, septal and hippocampal cholecystokinin and substance P neuronal systems to stress, in the male rat

The effects of acute and subchronic stress upon discrete cholecystokinin (CCK) and Substance P (SP) neuronal systems have been studied. Adult male rats were exposed to foot-shock stress for periods of 2, 4, 10, 30 or 60 min, immediately following which they were decapitated; brains were rapidly removed and frozen, and subsequently microdissected and extracted. CCK and SP were determined by RIA. In the olfactory tubercule, stress had no effect upon CCK content, but induced a rapid depletion of SP. In the prefrontal cortex, increased CCK concentrations were found following 30 min of stress exposure. In the medial septum, foot-shock led to a rapid increase in CCK content, and to a similar but delayed change in SP levels. A rapid rise in CCK concentrations was also seen in the lateral septum, but no stress effect whatsoever upon SP occurred in this structure. In the dentate gyrus, CCK exhibited a biphasic responsiveness to stress, while SP levels were increased only at the later time intervals. These data demonstrate that discrete CCK and SP neuronal systems are responsive to stress, and thereby support a functional role for these peptides in the processing of neural and hormonal signals by the CNS.     

Stress causes decrease in vascular relaxation linked with altered phosphorylation of heat shock proteins

Cyclic nucleotide-dependent vascular relaxation is associated with increases in the phosphorylation of a small heat shock protein (HSP), HSP20. An increase in phosphorylation of another small HSP, HSP27, is associated with impaired cyclic nucleotide-dependent vascular relaxation. Expression of HSPs is altered by exposure to several types of cellular stress in vitro. To determine if behavioral stress in vivo alters vascular expression and phosphorylation of the small HSPs and cyclic nucleotide-dependent vascular relaxation, borderline hypertensive rats were stressed by restraint and exposure to air-jet stress 2 h/day for 10 days or remained in their home cage. Stress impaired relaxation of aorta to forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This was associated with an increase in the aortic expression and phosphorylation of HSP27, which was localized to the vascular smooth muscle, but a decrease in the amount of phosphorylated (P)-HSP20. To determine if P-HSP27 inhibits phosphorylation of HSP20, P-HSP27 was added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of cAMP-dependent protein kinase. P-HSP27 inhibited phosphorylation of HSP20 in a concentration-dependent manner. These data demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. The increase in P-HSP27 and decrease in P-HSP20 were associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress in vivo, an effect similar to that observed previously in response to cellular stress in vitro.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme

A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to benzoyl-CoA. The further downstream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl-CoA is oxidized to 2,3-dihydro-2,3-dihydroxybenzoyl-CoA (benzoyl-CoA dihydrodiol) by benzoyl-CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxC(mal)) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3-dihydro-2,3-dihydroxybenzoyl-CoA + H(2)O --> 3,4-dehydroadipyl-CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring-(13)C(6)]benzoyl-CoA, benzoyl-CoA oxygenase/reductase, BoxC(mal) protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde; the missing one-carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde and [2,3,4,5,6-(13)C(5)]4,5-dehydroadipyl-CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed benzoyl-CoA-dihydrodiol lyase. It contains domains characteristic for enoyl-CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring-cleavage reaction does not require molecular oxygen.     

Influence of protein transduction domains on target-specific chimeric proteins

Direct targeting to the cytoplasm and nucleus using protein transduction domains (PTD) has been described to be efficient but non-cell-type-specific, and only has clinical relevance when the molecule is active exclusively in the diseased cell. The use of PTDs is an attractive mechanism to improve drug delivery. In this work, we designed recombinant proteins that contain epidermal growth factor as ligand to render uptake target cell-specific. We evaluated the potential of several PTDs to induce the cytosolic uptake of the catalytic domain of diphtheria toxin by measuring cytotoxicity. Although PTD-dependent membrane transfer is very low, the proteins exhibited concentration-dependent cytotoxic activity. Higher binding at 4 degrees C compared to 37 degrees C suggests that uptake by the PTDs MTS and TLM occurs via an endocytic pathway. Non-specific binding is predominantly a function of the PTD and greatly increases by substitution of a non-polar glycine with a negatively charged glutamate in the PTD HA2.     

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.     

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta . Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo . Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.     

The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.