The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor

Triosephosphate isomerase (TIM) has a conserved salt bridge 20 A away from both the active site and the dimer interface. In this study, four salt bridge mutants of Trypanosoma brucei brucei TIM were characterized. The folding and stability of the mutants are impaired compared to the wild-type enzyme. This salt bridge is part of a hydrogen bonding network which tethers the C-terminal beta7alpha7beta8alpha8 unit to the bulk of the protein. In the variants D227N, D227A, and R191S, this network is preserved, as can be deduced from the structure of the R191S variant. In the R191A variant, the side chain at position 191 cannot contribute to this network. Also the catalytic power of this variant is most affected.     

Oxyresveratrol and hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of action

Tyrosinase is responsible for the molting process in insects, undesirable browning of fruits and vegetables, and coloring of skin, hair, and eyes in animals. To clarify the mechanism of the depigmenting property of hydroxystilbene compounds, inhibitory actions of oxyresveratrol and its analogs on tyrosinases from mushroom and murine melanoma B-16 have been elucidated in this study. Oxyresveratrol showed potent inhibitory effect with an IC(50) value of 1.2 microm on mushroom tyrosinase activity, which was 32-fold stronger inhibition than kojic acid, a depigmenting agent used as the cosmetic material with skin-whitening effect and the medical agent for hyperpigmentation disorders. Hydroxystilbene compounds of resveratrol, 3,5-dihydroxy-4'-methoxystilbene, and rhapontigenin also showed more than 50% inhibition at 100 microm on mushroom tyrosinase activity, but other methylated or glycosylated hydroxystilbenes of 3,4'-dimethoxy-5-hydroxystilbene, trimethylresveratrol, piceid, and rhaponticin did not inhibit significantly. None of the hydroxystilbene compounds except oxyresveratrol exhibited more than 50% inhibition at 100 microm on l-tyrosine oxidation by murine tyrosinase activity; oxyresveratrol showed an IC(50) value of 52.7 microm on the enzyme activity. The kinetics and mechanism for inhibition of mushroom tyrosinase exhibited the reversibility of oxyresveratrol as a noncompetitive inhibitor with l-tyrosine as the substrate. The interaction between oxyresveratrol and tyrosinase exhibited a high affinity reflected in a K(i) value of 3.2-4.2 x 10(-7) m. Oxyresveratrol did not affect the promoter activity of the tyrosinase gene in murine melanoma B-16 at 10 and 100 microm. Therefore, the depigmenting effect of oxyresveratrol works through reversible inhibition of tyrosinase activity rather than suppression of the expression and synthesis of the enzyme. The number and position of hydroxy substituents seem to play an important role in the inhibitory effects of hydroxystilbene compounds on tyrosinase activity.     

Evolutionary relationship between different subgroups of restriction endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.     

A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.     

Insight into the kinetics and the mode of the interaction between smooth muscle calponin and F-actin

Kinetics of the smooth muscle calponin-F-actin interaction was studied by stopped-flow measurements of light scattering and fluorescence intensity of pyrene-labelled F-actin. The intensity and character of the changes in light scattering, and thus the mode of calponin binding to actin filaments leading to changes in their shape and bundling, depend on the molar ratio of the two proteins. Parallel measurements of pyrene-fluorescence quenching upon calponin binding revealed that intrinsic conformational changes in actin filaments are delayed relative to the binding process and are not markedly influenced by the mode of calponin binding. Bundling of actin filaments by calponin was not correlated with fluorescence changes and thus with alterations in the structure of actin filaments.     

Potassium-controlled synthesis of heterotopic macrocycles based on isothiosemicarbazide

The macrocyclisation reaction of 3,3′-(3,6-dioxaoctane-1,8-diyldioxy)-bis(2-hydroxybenzaldehyde) (1) with S-methylisothiosemicarbazide hydroiodide (H₂NNC(SCH₃)NH₂·HI) in the presence of potassium triflate, followed by addition of M(CH₃COO)₂·nH₂O, where M=Ni, Cu, Zn, afforded [NiLKI₃] (2), [NiLK(CF₃SO₃)] (3), [CuLK(CF₃SO₃)(CH₃OH)] (4) and [(ZnILK)₂CH₃OH] (5), respectively. Compounds 2-5 have been characterised by X-ray crystallography. IR, electronic, mass, ¹H, ¹³C{¹H} and ¹⁹F{¹H} NMR spectra are reported. Magnetic susceptibility measurements and ESR spectra of 4 indicate weak intermolecular spin-spin interactions, which are mostly dipolar in origin.     

Extrachromosomal rDNA amplification in the oocytes of Polystoechotes punctatus (Fabricius) (Insecta-Neuroptera-Polystoechotidae)

The ovary of Polystoechotes punctatus consists of several ovarioles of meroistic-polytrophic type. Histological, histochemical and ultrastructural studies revealed that the extrachromosomal amplification of rDNA takes place in the oocyte nucleus. Prior to previtellogenic growth the oocyte nucleus contains the chromosomes of meiotic prophase and a condensed extra DNA body. Initial split of extrachromosomal DNA material into several fragments coincides with the appearance of a spherical, fine granular body (referred to as primary nucleolus). Its gradual fragmentation accompanied by further dispersion of amplified DNA results in the formation of a growing number of multiple nucleoli. Until mid previtellogenesis each multiple nucleolus contains detectable amount of rDNA. In the advanced stages of previtellogenesis rDNA can hardly be visualized within the multiple nucleoli, while chromosomes form a few dense aggregates randomly disposed in the karyoplasm. At the onset of vitellogenesis the chromosomes assemble to form a karyosome. In its close vicinity DNA-positive material reaggregates. Multiple nucleoli are either found on the periphery of this aggregation or merge within it. At the final stages of vitellogenesis the number of multiple nucleoli significantly decreases.     

RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

Background  

Escherichia coli guanine-N2 (m²G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.