Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.     

Influence of estradiol and progesterone on the sensitivity of rat thoracic aorta to noradrenaline

The aim of this study was to investigate the effects of low and high doses of estradiol, and of progesterone on the response to noradrenaline in rat thoracic aorta. Two weeks after bilateral ovariectomy, female rats received a s.c. injection of vehicle (corn oil, 0.1 mL/day), estradiol (10 microg/kg/day or 4 mg/kg/day) and/or progesterone (20 mg/kg/day), for eight days. On the ninth day, the rats were sacrificed and aortic rings, with or without endothelium, were used to generate concentration-response curves to noradrenaline. Aortic rings with intact endothelium from the high-dose (4 mg/kg/day) estradiol group were supersensitive to noradrenaline compared to the vehicle or low-dose (10 microg/kg/day) estradiol groups (pD2 values = 7.86+/-0.09, 7.30+/-0.11 and 7.35+/-0.04, respectively). Endothelium-intact aortic rings from high-estradiol rats were supersensitive to noradrenaline when compared to vehicle-, progesterone- and progesterone + high-estradiol-treated rats (pD2 values = 7.77+/-0.12, 7.21+/-0.13, 6.93+/-0.04 and 7.22+/-0.18, respectively). There were no significant differences among the pD2 values for noradrenaline in aortic rings without endothelium. In conclusion, at high but not low doses, estradiol increased the sensitivity to noradrenaline and this was prevented by progesterone. Both of these effects were endothelium-dependent.     

Wound-inducible proteinase inhibitors in pepper. Differential regulation upon wounding, systemin, and methyl jasmonate

Seven small (approximately 6,000 D) wound-inducible proteinase inhibitor proteins were isolated from leaves of pepper (Capsicum annuum) plants that are members of the potato inhibitor II family. N-terminal sequences obtained indicated that the pepper leaf proteinase inhibitors (PLPIs) exhibit homology to two GenBank accessions that code for preproteins containing three isoinhibitors domains each that, when post-translationally processed, can account for the mixture of isoinhibitors that are reported herein from pepper leaves. A constitutive level of PLPI proteins was found in pepper leaves, and these levels increased up to 2.6-fold upon wounding of the lower leaves. Exposing intact plants to methyl jasmonate vapors induced the accumulation of PLPIs. Supplying excised young pepper plants with water through the cut stems induced PLPI proteins to levels higher than those found in intact plants, but with high variability. Supplying the excised plants with systemin did not result in an increase of PLPI levels that were statistically higher than levels found in excised plants. Gel-blot analyses of PLPI induction revealed the presence of two mRNA bands, having slightly different mobilities in agarose gels. Only the low M(r) mRNA is present in untreated control plants, and it appears to be responsible for the constitutive levels of PLPI found in leaves. Both mRNA species are wound- and methyl jasmonate-inducible. Only the low- M(r) species is weakly induced by systemin, indicating a differential expression of the two PLPI species.     

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both M?ssbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.     

Identification of Cryptosporidium felis in a cow by morphologic and molecular methods

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 +/- 0.4 micrometer; range, 3.5 to 5.0 micrometer), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.     

Incorporation of either molybdenum or tungsten into formate dehydrogenase from<Emphasis Type="Italic"> Desulfovibrio alaskensis</Emphasis> NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, =93±3 kDa and =32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5–40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d¹ configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.Abbreviations AOR aldehyde oxidoreductase - FDH formate dehydrogenase - NAP periplasmic nitrate reductase - SRB sulfate-reducing bacteria     

Reactive phenotypes after acute and chronic NK-cell activation

Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules that have been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as target cell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cell activation.     

Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas chlororaphis strains DSM 50083T and DSM 50135

Two azurins (Az624 and Az626) were isolated from the soluble extract of two strains of Pseudomonas chlororaphis, DSM 50083(T) and DSM 50135, respectively, grown under microaerobic conditions with nitrate as final electron acceptor. The azurins, purified to electrophoretic homogeneity in three chromatographic steps, exhibit several peculiar properties. They have high reduction potentials and lower pI than most azurins described in the literature. As previously observed for Pseudomonas aeruginosa azurin, their reduction potentials are pH-dependent, but the pK values of their oxidized forms are lower, which suggests that deeper structural changes are associated with the oxidation process of these novel azurins. A hitherto undescribed pH-dependence of the diffusion coefficient was observed in Az624, that could be caused either by conformational changes, or by the formation of supramolecular aggregates associated with a protonation process. Both azurins exhibit axial X-band electron paramagnetic resonance spectra in frozen solution showing a typical hyperfine with the copper nucleus (I=3/2) and a well-resolved superhyperfine structure with two equivalent 14N nucleus (I=1), which is not usually observed for azurins from other species.