The characterization of the exposure to immune mediated apoptosis and the regulation of immune cytotoxic activity in the environment of a neoplasm and in decidua

Acquiring the immune-mediated apoptosis and the ability to regulate the cytotoxic immune response are the main phenomena playing fundamental roles in such situations as neoplasm survival and creation of immune tolerance during pregnancy. The aim of this study was to investigate these phenomena through the evaluation of metallothionein and RCAS1 proteins in neoplasm and its healthy environment (clear surgical margin), physiological conditions in placenta and its environment (decidua) and the comparison to non-neoplasmatic lesions originating from the environment (nasal polyps, endometriosis). We have shown that the growth of RCAS1 expression was simultaneous to the infiltration of activated immunological cells of tumor environment as well as decidua. The activity of immunological cells was in our study selectively suppressed. Metallothionein expression growth was also observed in healthy tumors stroma and in decidua probably in response to the growing cytotoxic activity and tumor spread. Alterations in RCAS1 and Metallothionein expression seem to be associated with local immune dysfunction in nasal polyps and endometriosis. In conclusion, the ability to compensate the growing cytotoxic immune response is physiologically observed in decidua, the lost of this ability in tumor environment might participate in the development of tumor spread.     

Pharmacological profile of the "triple" monoamine neurotransmitter uptake inhibitor, DOV 102,677

1. The molecular and behavioral pharmacology of DOV 102,677 is characterized. 2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, "triple" uptake inhibitor that suppresses [(3)H]dopamine (DA), [(3)H]norepinephrine (NE) and [(3)H]serotonin (5-HT) uptake by recombinant human transporters with IC(50) values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k (i) values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of beta-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days). 4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity. 5. In summary, DOV 102,677 is an orally active, "balanced" inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.     

Pharmacological Profile of the “Triple” Monoamine Neurotransmitter Uptake Inhibitor, DOV 102,677

Summary 1. The molecular and behavioral pharmacology of DOV 102,677 is characterized.2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, “triple” uptake inhibitor that suppresses [³H]dopamine (DA), [³H]norepinephrine (NE) and [³H]serotonin (5-HT) uptake by recombinant human transporters with IC₅₀ values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k _i values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of β-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days).4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity.5. In summary, DOV 102,677 is an orally active, “balanced” inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.     

Spontaneous apoptosis of melanotic and amelanotic melanoma cells in different phases of cell cycle: relation to tumor growth

Since the spontaneous alteration of native melanotic (Ma) into amelanotic (Ab) transplantable melanoma line it has been observed that this alteration is accompanied by the acceleration of growth of Ab line. The aim of the present study was to check and estimate spontaneous apoptosis of cells from cell cycle phases. Cytometric cell cycle analysis was performed by staining cells with propidium iodide (PI). Apoptosis estimated by the TUNEL method, alterations in the plasma membrane structure (annexin V staining), changes in the mitochondrial transmembrane potential--delta psi m (JC-1 staining) showed that amelanotic melanoma cells have decreased ability to undergo spontaneous apoptosis. The obtained results showing that in the native melanotic line about 30% of cells are in S+G2/M phases and that 33% of these cells undergo apoptosis could lead to the conclusion that the slower growth of this melanoma line is the result of lower proliferation activity and higher rate of apoptosis of these tumor cells. The number of cells in S+G2/M phases in amelanotic melanoma line increases up to 40% and only 7% of them undergo apoptosis. This observation seems to suggest that the expansive growth of this melanoma line depends mainly on the decreased ability to undergo spontaneous apoptosis, especially in case of cells from S+G2/M phases. Moreover, the obtained results indicate that alteration of melanotic line into amelanotic one, accompanied by differences in many biological features also concerns basic cell processes such as cell cycle and cell death.     

Sequence of deltaF508 CFTR allele identified at present is lacking in medieval specimens from Central Poland. Preliminary results

deltaF508 is the most common (70%) among over 1000 mutations of the gene encoding ATP-regulated chloride channel, namely CFTR--cystic fibrosis transmembrane regulator. The time which passed from the calculated mutation event was anticipated on the basis of the frequency of contemporary haplotypes, but not on its direct identification. The presence of three base pairs deletion in the ancient DNA (aDNA) isolated from skeletal remains of the Middle Ages origin was investigated. Teeth excavated in the area of three sites located in Central Poland were processed for a DNA. 6 out of 82 samples did not produce amplificable fragments of DNA. Although the number of specimens analyzed was sufficient to confirm the presence of the rare mutation, the deltaF508 CFTR sequence was not found in the remains of individuals living back 35 - 45 generations. The absence of the mutated allele in the particular geographic region cannot state for the status of mutated allele throughout the country, especially at times when migrations were limited and movements of people were more area restricted than at present days.     

Phosphate positioning and availability in the starch granule matrix as studied by EPR

Cu(2+) was introduced as an EPR probe into the starch granules isolated from different starch crop genotypes including transgenically modified potatoes generated for extreme amylose and starch phosphate monoester concentrations. Several discrete copper adducts bound to the starch matrix with different strength was revealed. It was found that phosphate has a significant influence on the type of these species, their number, location in the structure, and strength of binding. Well dispersed Cu(2+) complexes with axial symmetry are formed in the semicrystalline part of the starch linked through O-P- bonds in the phosphorylated starches. In the amorphous part of the starch, freely rotating hexaaqua complexes of Cu(2+) and complexes coupled antiferromagnetically are formed. The amount of the former increases with content of phosphate indicating enhanced binding of water in the granules. The results complement previous experimental data and molecular models for the starch granule with respect to the location and effects of phosphate and crystalline matter.     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

The use of <Emphasis Type="Italic">spa</Emphasis> and phage typing for characterization of clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in the University Clinical Center in Gdańsk,Poland

The emergence of spa types and spa–clonal complexes (CC) among clinical methicillin-resistant Staphylococcus aureus isolates collected from the University Clinical Center in Gdańsk between 2008 and 2009 were investigated. Phage typing was used as the initial screening in the study. The basic set of phages and the additional set of phages were used. Most of the isolates (56 %) belonged to the phage group III. With the additional set of phages, eight types were found, with predominant one MR8 (50 %). Sixteen distinct spa types were observed. The most frequent were t003 (22 %), t151 (16 %), and t008 (12 %). The spa types were clustered into two spa-CC and eight singletons. The predominant CC010 (50 %) consisted of six types, with the most common t003 (36.7 %) and t151(26.7 %), and in 80 % was identified as staphylococcal chromosomal casette mec (SCCmec) type II. The second cluster has no founder (12 %) with only two spa types: t037 belonging to SCCmec type III and t029. In the most frequent singleton, spa type t008 alone was clustered in 12 % of the isolates. All singletons correspond to SCCmec type IV. The CC010 was distributed in most of the hospital wards, corresponded to Multilocus sequence typing type ST5/ST225 and was constantly present throughout the observed period. The isolates of CC010 generally belonged to the phage group III, and most of them (53.3 %) were resistant to erythromycin, clindamycin, and ciprofloxacin. The concordance between spa-clone and phage type was very high, but the same phage type MR8 was observed within different spa types of the predominant clone.     

Short‐term exposure to 50 Hz ELF‐EMF alters the cisplatin‐induced oxidative response in AT478 murine squamous cell carcinoma cells

The aim of this study was to assess the influence of cisplatin and an extremely low frequency electromagnetic field (ELF‐EMF) on antioxidant enzyme activity and the lipid peroxidation ratio, as well as the level of DNA damage and reactive oxygen species (ROS) production in AT478 carcinoma cells. Cells were cultured for 24 and 72 h in culture medium with cisplatin. Additionally, the cells were irradiated with 50 Hz/1 mT ELF‐EMF for 16 min using a solenoid as a source of the ELF‐EMF. The amount of ROS, superoxide dismutase (SOD) isoenzyme activity, glutathione peroxidase (GSH‐Px) activity, DNA damage, and malondialdehyde (MDA) levels were assessed. Cells that were exposed to cisplatin exhibited a significant increase in ROS and antioxidant enzyme activity. The addition of ELF‐EMF exposure to cisplatin treatment resulted in decreased ROS levels and antioxidant enzyme activity. A significant reduction in MDA concentrations was observed in all of the study groups, with the greatest decrease associated with treatment by both cisplatin and ELF‐EMF. Cisplatin induced the most severe DNA damage; however, when cells were also irradiated with ELF‐EMF, less DNA damage occurred. Exposure to ELF‐EMF alone resulted in an increase in DNA damage compared to control cells. ELF‐EMF lessened the effects of oxidative stress and DNA damage that were induced by cisplatin; however, ELF‐EMF alone was a mild oxidative stressor and DNA damage inducer. We speculate that ELF‐EMF exerts differential effects depending on the exogenous conditions. This information may be of value for appraising the pathophysiologic consequences of exposure to ELF‐EMF. Bioelectromagnetics 33:641–651, 2012. © 2012 Wiley Periodicals, Inc.