Properties of monovalent nickel complexes with tetraaza-macrocyclic ligands in aqueous solutions

The effects of N-alkylation on the redox potential of the couples NiLⁱ²⁺/NiLⁱ⁺, L = tetraaza-14-membered-macrocyclic ligands, and on the properties of the monovalent nickel complexes in aqueous solutions are reported for 14 complexes. The spectra and lifetimes of the NiLⁱ⁺ complexes are reported. The self-exchange rates for the couples NiLⁱ²⁺/NiLⁱ⁺ were determined. Two of the ligands were synthesized for the first time for this study. Cyclic voltammetry and pulse radiolysis were used. The results point out that: (i) N-alkylation always shifts the redox potential to a less cathodic one; this effect stems to a large degree from the decrease in the solvation energy of the complex caused by the N-alkylation of the ligand. (ii). The lifetime of the monovalent complexes is not linearly related to the redox potential of the NiLⁱ²⁺/NiLⁱ⁺ couples. (iii) The NiLⁱ⁺ complexes exist in several isomeric forms; the rate of the isomerization depends on the structure of the ligand. (iv) Different isomers of NiLⁱ⁺ may be formed when the complex NiLⁱ²⁺ is reduced by different reagents; therefore, the pulse-radiolytically formed NiLⁱ⁺ complexes might have different properties than those formed electrochemically.     

A Point Mutation in Translation Initiation Factor 2B Leads to a Continuous Hyper Stress State in Oligodendroglial-Derived Cells

Background

Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM). The disease is manifested by loss of brain myelin upon physiological stress. In a previous study, we showed that fibroblasts isolated from CACH/VWM patients are hypersensitive to pharmacologically-induced endoplasmic reticulum (ER) stress. Since brain cells from affected individuals are not available for research, we wished to assess the effect of eIF2B mutation on oligodendroglial-derived cells.

Methodology/Principal Findings

A rat oligodendroglial-derived cell line was used for a stable knock-down of eIF2B5 followed by stable expression of mutated eIF2B5(R195H) cDNA. In response to a pharmacological ER-stress agent, eIF2B5(R195H) expressing cells exhibited heightened ER-stress response demonstrated by hyper induction of ATF4, GADD34, Bip, PDIA1, PDIA3, PDIA4 and PDIA6 proteins. Moreover, even in the absence of a pharmacological stress agent, eIF2B5(R195H)-expressing cells exhibited high basal levels of ATF4, GADD34 and ER-associated Bip, PDIA1 and PDIA3.

Significance

The data provide evidence that oligodendroglial-derived cells expressing a mutated eIF2B constantly use their stress response mechanism as an adaptation mean in order to survive. The current study is the first to demonstrate the effects of eIF2B5 mutation on ER homeostasis in oligodendroglial-derived cells.     

Synthesis and Anticancer Activity of Functionalized Thieno[2,3-d]pyrimidine Compounds and Their Triazinyl and Tetrazinyl Derivatives

Russian Journal of Bioorganic Chemistry - New thienopyrimidine derivatives substituted with amino and hydrazinyl side chains were synthesized starting with 2-hydrazinyl substituted thienopyrimidine...     

Truncated vitronectins: binding to immobilized fibrin and to fibrin clots, and their subsequent interaction with cells.

The plasminogen activator inhibitor-1 (PAI-1) is stabilized in its inhibitory conformation by binding to Vitronectin (Vn). The anchorage of PAI-1 to the fibrin fibers was recently shown to be mediated by Vn, and as such to modulate fibrinolysis. Here we report the mapping of the fibrin binding sites in Vn using truncated recombinant Vns, and show that two segments of Vn are involved: one at its carboxyl terminus (within residues 348-459) and one at its amino terminus (within residues 1-44). This mapping sets the stage for (i) the design of specific inhibitors for the Vn-fibrin interaction; (ii) for studying the role of this interaction in the anchoring of endothelial cells and platelets onto the fibrin clot; and (iii) for getting a deeper insight into the mechanism of the Vn-fibrin interaction in fibrinolysis. (c)2002 Elsevier Science.     

New quinoxaline 1,4-di-N-oxides. Part 1: Hypoxia-selective cytotoxins and anticancer agents derived from quinoxaline 1,4-di-N-oxides

Hypoxic cells which are common feature of solid tumors are resistant to both anticancer drugs and radiation therapy. Thus, the identification of drugs with the selective toxicity toward hypoxic cells is an important target in anticancer chemotherapy. Tirapazamine has been shown to be an efficient and selective cytotoxin after bioreductive activation in hypoxic cells which is thought to be due to the presence of the 1,4-di-N-oxide. A new series of quinoxaline 1,4-di-N-oxides and fused quinoxaline di-N-oxides were synthesized and evaluated for hypoxic-cytotoxic activity on EAC cell line. Compound 10a was the most potent cytotoxin IC(50) 0.9 microg/mL, potency 75 microg/mL, and was approximately 15 times more selective cytotoxin (HCR>111) than 3-aminoquinoxaline-2-carbonitrile which has been used as a standard (HCR>7.5). Compounds 4 and 3a,b were more selective than the standard. In addition, antitumor activity against Hepg2 (liver) and U251 (brain) human cell lines was evaluated, compounds 9c and 8a were the most active against Hepg2 with IC(50) values 1.9 and 2.9 microg/mL, respectively, however, all the tested compounds were nontoxic against U251 cell line.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.     

Nitrogen-induced variations in leaf gas exchange of spring triticale under field conditions

Nitrogen (N) is the key factor limiting photosynthetic processes and crop yield. Little is known about the response of leaf gas exchange of spring triticale (Triticosecale Wittm.) to N supply. The effect of N fertilizers on different gas exchange variables, i.e., photosynthetic rate (A), transpiration rate (E), stomatal conductance (g _s), instantaneous water use efficiency (WUE) and maximum quantum yield of photosystem II (PSII) (F _v/F _m), chlorophyll index (SPAD, soil–plant analysis development), and the relationship of these variables with yield were studied in spring triticale grown under field conditions. Six treatments of N—0, 90, 180, 90 + 30, 90 + 30 + 30 kg ha^?1 (applied as ammonium nitrate, AN) and one treatment of N 90 + 30 + 30 kg ha^?1 (applied as urea ammonium nitrate solution, UAN) were compared. The analysis of variance showed that throughout the triticale growing season, N fertilization had significant effects on A, WUE, g _s and SPAD. On average, N fertilizer application increased A values by 14–70%. E and F _v/F _m values were not influenced by N fertilization levels. The effect of growth stage and year on gas exchange variables and F _v/F _m and SPAD was found to be significant. At different growth stages, A values varied and maximum ones were reached at BBCH 31–33 (decimal code system of growth stages) and BBCH 59. With aging, values of A decreased independently of N fertilization level. The gas exchange variables were equally affected by both fertilizer forms. The interplay among grain yield, leaf gas exchange variables, F _v/F _m and SPAD of spring triticale was estimated. The statistical analysis showed that grain yield positively and significantly correlated with A and SPAD values throughout the growing season.