Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

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.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Preferential expression of transient potassium current (IA) by 'short' hair cells of the chick's cochlea

We have made a comparative study of the membrane properties of tall and short hair cells isolated from a selected region of the chick's cochlea. Tall hair cells are analogous to inner cochlear hair cells of mammals, and like those, are presynaptic to the majority of afferent neurons in the cochlea. Short hair cells, like mammalian outer hair cells, are the postsynaptic targets of efferent neurons that inhibit the cochlea. Voltage-clamp recordings have revealed that short hair cells have an inactivating potassium (K) current, IA, whereas tall hair cells have little or none. Short hair cells are also sensitive to the cholinergic agonist carbachol, whereas tall hair cells are not. This pattern is in accord with the selective distribution of efferent cholinergic synapses in the cochlea. Although IA is completely inactivated at the resting potential of the short hair cells, cholinergic agonists can hyperpolarize these cells by as much as 30 mV. This hyperpolarization removes inactivation and allows IA to modulate subsequent voltage-dependent processes in short hair cells. It is concluded that IA could increase the high frequency response of the hair cell by decreasing membrane resistance and thus the membrane time constant after inhibition. This will be of particular importance to cochlear function if short hair cells produce voltage-dependent movements, as do mammalian outer hair cells.     

Influence of interferon-gamma and extracellular tryptophan on indoleamine 2,3-dioxygenase activity in T24 cells as determined by a non-radiometric assay.

The indoleamine 2,3-dioxygenase (EC 1.13.11.17) activity in human T24 cells has been investigated in cell extracts by using a non-radioactive assay. It is enhanced in a dose-dependent manner up to 25-fold by interferon-gamma. The maximum reaction velocity is increased rather than the Km, which remains at 4 mumol/l. Induction of activity starts 3 h after stimulation and reaches a plateau at 21-48 h. Decreased stimulation was observed in the presence of high L-tryptophan concentrations.     

A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.