Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.     

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua.

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.     

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

The dependence of the frequency of recessive lethal and vital mutations inArabidopsis thaliana (L.)Heynh. on the concentration of N-nitroso-N-methylurea

The dependence of the frequency of recessive lethal (two groups), chlorophyll and morphological mutations on the mutagen concentration was determined in M₂ after subjection to N-nitroso-N-methylurea applied to seeds ofArabidopsis in three concentrations (0·05, 0·10 and 0·20mm). The observed frequencies were compared with the theoretically expected ones for the linear and for two exponential types of dependence, by using the t-test, according to the formulas m=k. C, m=k. C^3/2, m=k. C². No satisfactory agreement with any expected type of dependence was found when directly observed frequencies were used. Since a considerable deficit of mutation frequency was observed in high concentrations, the correction of frequency values was done with respect to the probability of occurence of double mutations. After such a correction, a clear exponential relation was found in both types of lethals and a linear one in chlorophyll and morphological mutations. The probable occurence of multiple mutations should be, therefore, taken into account if the dependence of mutation frequency on the concentration of mutagen is discussed.     

Leaf Factors Affecting Light-Saturated Photosynthesis in Ecotypes of Solidago virgaurea from Exposed and Shaded Habitats

Plants of Solidago virgaurea L. from exposed and shaded habitats differ with respect to the response of the photosynthetic apparatus to the level of irradiance during growth. An analysis was carried out on leaf characteristies which might be responsible for the differences established in the rates of Hght-saturated CO₂ uptake. The clones were grown in controlled environment chambers at high and low levels of irradiance. Light-saturated rates of photosynthesis and transpiration were measured at natural and lower ambient CO₂ concentrations. A low temperature dependence of light-saturated CO₂ uptake at natural CO₂ concentrations, and a strong response to changes in stomatal width, suggested that the rate of CO₂ transfer from ambient air towards reaetion sites in chloroplasts was mainly limiting the pholosynthetic rate. Resistances to transfer of CO₂ for different parts of the pathway were calculated. There was a weak but significant correlation between stomatal conductance and the product stomatal frequency ± pore length. Mesopbyll conductance and dry weight per unit area were highly correlated in leaves not damaged by high irradiance. This suggests that mesophyll conductance increases with increasing cross sectional area (per unit leaf area) of the pathways of CO₂ transfer in the mesophyll from cell surfaces to reaction sites. The higher light-saturated photosynthesis in clones from exposed habitats when grown at high irradiance than when grown at low irradiance was attributable mainly to a lower mesophyll resistance. In shade clones the effect upon CO₂ uptake of the increase in leaf thickness when grown at high irradiance was counteracted by the associated inactivation of the photosynthetic apparatus. The difference in CO₂ uptake present between clones from exposed and shaded habitats when preconditioned to high irradiance resulted from differences in both mesophyll and stomatal resistances. A few hybrid clones of an F₁-population from a cross between a clone from an exposed habitat and a clone from a shaded habitat reacted, on the whole, in the same way as the exposed habitat parent. When grown at high irradiance, the hybrid clones showed higher photosynthetic rates than either parent; this was largely attributable to the unusually low stomatal resistance of the hybrid leaves.     

Synthesis and evaluation of bifunctional tetrahydroxamate chelators for labeling antibodies with 89Zr for imaging with positron emission tomography

Two novel bifunctional tetrahydroxamate chelators 3 and 4 were synthesized and evaluated for labeling antibodies with ⁸⁹Zr for positron emission tomography imaging. Compared to previously reported tetrahydroxamate chelators 1 and 2 with an iminodiacetamide backbone, 3 and 4 were based on an extended iminodipropionamide and dipropylenetriamine backbone, respectively. Trastuzumab conjugates of 3 and 4 were efficiently labeled with ⁸⁹Zr (>95% radiochemical yield). The in vitro plasma stability of ⁸⁹Zr-4-Trastuzumab and especially ⁸⁹Zr-3-Trastuzumab was greatly improved over previously reported ⁸⁹Zr-1-Trastuzumab and ⁸⁹Zr-2-Trastuzumab, but their demetalation remained higher and faster than ⁸⁹Zr-deferoxamine (DFO)-Trastuzumab. These observations were confirmed by PET imaging and biodistribution in mice, with significant higher bone uptake for ⁸⁹Zr-4-Trastuzumab, followed by ⁸⁹Zr-3-Trastuzumab, and to a lesser extent for ⁸⁹Zr-DFO-Trastuzumab. Molecular modeling showed that 3 and 4 with an extended backbone could form eight-coordinate Zr-complexes as compared to only seven-coordinate Zr-complexes of 1 and 2. Our data suggest further elongation of linker length between hydroxamate motifs of this class of chelators is needed to reach a better Zr-coordination configuration and improve in vivo stability.     

Structural design,synthesis and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents

4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.     

Characterization of As(III) oxidizing <Emphasis Type="Italic">Achromobacter</Emphasis> sp. strain N2: effects on arsenic toxicity and translocation in rice

Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L^?1 of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils.