shRNA silencing of AS3MT expression minimizes arsenic methylation capacity of HepG2 cells

Several methyltransferases have been shown to catalyze the oxidative methylation of inorganic arsenic (iAs) in mammalian species. However, the relative contributions of these enzymes to the overall capacity of cells to methylate iAs have not been characterized. Arsenic (+3 oxidation state) methyltransferase (AS3MT) that is expressed in rat and human hepatocytes catalyzes the conversion of iAs, yielding methylated metabolites that contain arsenic in +3 or +5 oxidation states. This study used short hairpin RNA (shRNA) to knock down AS3MT expression in human hepatocellular carcinoma (HepG2) cells. In a stable clonal HepG2/A cell line, AS3MT mRNA and protein levels were reduced by 83 and 88%, respectively. In comparison, the capacity to methylate iAs decreased only by 70%. These data suggest that AS3MT is the major enzyme in this pathway, although an AS3MT-independent process may contribute to iAs methylation in human hepatic cells.     

Immunological problems of polymer-bound drugs

For the application of polymer-bound drugs (especially in combination with the targeting antibodies), it is essential to know more about the immunogenicity and possible hazards from immune reactions induced by their repeated application. The immune reaction depends on many factors (including structure of antigen, dose of antigen, schedule and way of the application, genetic background of the immunized, i.e., receiving organism) and could be weakened or eliminated by a choice of suitable factors. Problems connected with the immune reaction of the humoral and cellular type (B and T cell involvement) against different polymers and polymer-bound drugs and possible consequences for their clinical use will be discussed.     

The biphasic course of changes of left ventricular outflow gradient after alcohol septal ablation for hypertrophic obstructive cardiomyopathy

BACKGROUND AND AIM: Alcohol septal ablation (PTSMA) decreases left ventricular outflow gradient (LVOG) and relieves symptoms in patients with hypertrophic obstructive cardiomyopathy (HOCM). The time course of early changes of LVOG has not been clearly determined up to now. METHODS: Doppler echocardiography was used to determine the maximal LVOG. Thirty-nine consecutive patients (24 women; age 55+/-14) were examined at baseline, immediately after the procedure, and 3-5 days, 3 weeks and 3 months thereafter. RESULTS: The baseline LVOG decreased immediately after PTSMA from 73+/-49 mmHg to 13+/-16 mmHg (p<0.01). During the hospital stay (postprocedural period) LVOG increased from 13+/-16 to 37+/-35 mmHg (p<0.01). All the patients were discharged 5-10 days after the procedure. At three-week examination LVOG decreased from 37+/-35 to 25+/-12 mmHg (p<0.01). Three-month survival was 97%. One patient died suddenly one month after PTSMA. At three-month examination LVOG decreased from 25+/-12 to 17+/-14 mmHg (NS). All the patients reported an improvement in symptoms at follow-up. CONCLUSIONS: The immediate decrease of LVOG after PTSMA procedure caused by myocardial necrosis and stunning, was followed by a significant LVOG increase during the early postprocedural period and continuous LVOG decrease at the short-term follow-up.     

Splenectomy influences homing of transplanted stem cells in bone marrow-ablated mice

Cell mobilization, a process that influences circulation, margination, and finally, homing play key roles in the regeneration processes mediated by stem cells. Recent studies as well as prior studies from our group indicate an important role of the spleen in hematopoietic reconstitution, but to date the role of the spleen in hematopoietic reconstitution has been unclear and it has not been precisely documented in ablated animals. Therefore, we undertook the present study to define more closely the role of the spleen in hematopoietic reconstitution in lethally irradiated mice. After transplantation of irradiated mice with lacZ+ -marked lin- / CD117+ bone marrow cells, we compared splenectomized mice (T(S), splenectomy performed prior to irradiation) to nonsplenectomized, irradiated mice (T(N)) as well as to normal (unirradiated) mice. Impaired hematopoietic reconstitution was observed in T(S) mice. Splenectomy markedly altered the distribution of hematopoietic stem cells, as demonstrated by fluorescence-activated cell sorting analysis of endogenous CD117+ cells in the thymus and bone marrow of recipients. Cell engraftment was demonstrated by histochemical and polymerase chain reaction analyses of recipient tissues. These experiments demonstrated that in T(S) animals, transplanted hematopoietic stem cells mobilized to extravascular tissues, particularly the gastrointestinal tract. The number of donor cells in recipient tissues continued to increase for 30 days after transplantation with the highest numbers observed in the T(S) group. DNA marking analysis led to the conclusion that engrafted cells were not only integrated into recipient tissues but were also capable of performing complex cellular processes, including proliferation and repair. Our results are consistent with the novel possibility that cellular repair markedly affects stem cell regenerative functions and that repair is markedly influenced by the integrity and presence of organs not directly involved in specific tissue regeneration processes, particularly the spleen.     

Functional Magnetic Resonance Imaging in Patients with the Wet Form of Age-Related Macular Degeneration

The study is designed to determine the relationship between the progress of the wet form of age-related macular degeneration and the activity of the visual cortex examined using functional magnetic resonance imaging. Ten patients with the wet form of age-related macular degeneration (9 female and 1 male) with a mean age of 74.7 years (58–85 years) at various stages of bilateral involvement of the disease were included. Patients did not suffer from any other ocular nor neurological disease. All the patients underwent functional magnetic resonance imaging examinations with stimulation of both eyes using a black-and-white checkerboard of size 25.8 × 16.2 degrees. The group was compared with a group of healthy subjects with an average age of 54.1 years (45–65 years). For statistical evaluation, the Mann-Whitney U test was used. Comparing the extent of visual cortex activations we found a statistically significant difference between both the groups (p = 0.0247). However, the dependence of functional magnetic resonance imaging activity on visual acuity was not statistically significant (p = 0.223). We conclude that in patients with the wet form of age-related macular degeneration, lower functional magnetic resonance imaging activity of the visual cortex was found compared with the control group of healthy subjects. Dependence of functional magnetic resonance imaging activity on visual acuity was not statistically significant.     

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.     

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.     

Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca(2+)-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation-contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.