Reversible acetylation at the epsilon-amino group of lysines located at the conserved domain of core histones is supposed to play an important role in the regulation of chromatin structure and its transcriptional activity. One promising strategy for analyzing the precise function of histone acetylation is to block the activities of acetylating or deacetylating enzymes by specific inhibitors. Recently, two microbial metabolites, trichostatin A and trapoxin, were found to be potent inhibitors of histone deacetylases. Trichostatin A reversibly inhibits the mammalian histone deacetylase, whereas trapoxin causes inhibition through irreversible binding to the enzyme. The histone deacetylase from a trichostatin A-resistant cell line is resistant to trichostatin A, indicating that the enzyme is the primary target. Both of the agents induce a variety of biological responses of cells such as induction of differentiation and cell cycle arrest. Trichostatin A and trapoxin are useful in analyzing the role of histone acetylation in chromatin structure and function as well as in determining the genes whose activities are regulated by histone acetylation.

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Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methyl viologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Met150Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Induction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either N ω -nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO + ) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6-20 mg kg -1 ). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.

Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitro; conversely, it was remarkably reduced in vivo by pharmacological blockade of NOS. We conclude that HO-1 may function in antiapoptotic defense of the tumour, and thus it may have important protective and beneficial effects for tumour cells against oxidative stress induced by NO, which is produced in excess during solid tumour growth in vivo.

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A-factor, containing a gamma-butyrolactone in its structure, is an autoregulatory factor or a 'microbial hormone' controlling secondary metabolism and cellular differentiation in Streptomyces griseus. A-factor exerts its regulatory role by binding to a specific receptor protein which, in the absence of A-factor, acts as a repressor-type regulator for morphological and physiological differentiation. In the signal relay leading to streptomycin production in S. griseus, the A-factor signal is transferred from the A-factor receptor to the upstream activation sequence of a regulatory gene, strR, in the streptomycin biosynthetic gene cluster via an A-factor-dependent protein that serves as a transcription factor for strR. The StrR protein thus induced appears to activate the transcription of other streptomycin-production genes. The presence of A-factor homologues in a wide variety of Streptomyces species and distantly related bacteria implies the generality of gamma-butyrolactones as chemical cellular signalling molecules in microorganisms.