Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

The collective dimension of freedom of religion or belief in international law : the application of findings to the case of Turkey

Pertinent domestic and international developments involving issues related to tensions affecting religious or belief communities have been increasingly occupying the international law agenda. Those who generate and, thus, shape international law jurisprudence are in the process of seeking some of the answers to these questions. Thus the need for reconceptualization of the right to freedom of religion or belief continues as demands to the right to freedom of religion or belief challenge the boundaries of religious freedom in national and international law. This thesis aims to contribute to the process of “re-conceptualization” by exploring the notion of the collective dimension of freedom of religion or belief with a view to advance the protection of the right to freedom of religion or belief. The case of Turkey provides a useful test case where both the domestic legislation can be assessed against international standards, while at the same time lessons can be drawn for the improvement of the standard of international review of the protection of the collective dimension of freedom of religion or belief. The right to freedom of religion or belief, as enshrined in international human rights documents, is unique in its formulation in that it provides protection for the enjoyment of the rights “in community with others”.1 It cannot be realized in isolation; it crosses categories of human rights with aspects that are individual, aspects that can be effectively realized only in an organized community of individuals and aspects that belong to the field of economic, social and cultural rights such as those related to religious or moral education. This study centers on two primary questions; first, what is the scope and nature of protection afforded to the collective dimension of freedom of religion or belief in international law, and, secondly, how does the protection of the collective dimension of freedom of religion or belief in Turkey compare and contrast to international standards? Section I explores and examines the notion of the collective dimension of freedom of religion or belief, and the scope of its protection in international law with particular reference to the right to acquire legal personality and autonomy religious/belief communities. In Section II, the case study on Turkey constitutes the applied part of the thesis; here, the protection of the collective dimension is assessed with a view to evaluate the compliance of Turkish legislation and practice with international norms as well as seeking to identify how the standard of international review of the collective dimension of freedom of religion or belief can be improved.

Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80ºC until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Adventures of Ludom: a Videogame Geneontology

Within the last few decades, the videogame has become an important media, economic, and cultural phenomenon. Along with the phenomenon’s proliferation the aspects that constitute its identity have become more and more challenging to determine, however. The persistent surfacing of novel ludic forms continues to expand the conceptual range of ‘games’ and ‘videogames,’ which has already lead to anxious generalizations within academic as well as popular discourses. Such generalizations make it increasingly difficult to comprehend how the instances of this phenomenon actually work, which in turn generates pragmatic problems: the lack of an applicable identification of the videogame hinders its study, play, and everyday conceptualization. To counteract these problems this dissertation establishes a geneontological research methodology that enables the identification of the videogame in relation to its cultural surroundings. Videogames are theorized as ‘games,’ ‘puzzles,’ ‘stories,’ and ‘aesthetic artifacts’ (or ‘artworks’), which produces a geneontological sequence of the videogame as a singular species of culture, Artefactum ludus ludus, or ludom for short. According to this sequence, the videogame’s position as a ‘game’ in the historicized evolution of culture is mainly metaphorical, while at the same time its artifactuality, dynamic system structure, time-critical strategic input requirements and aporetically rhematic aesthetics allow it to be discovered as a conceptually stable but empirically transient uniexistential phenomenon that currently thrivesbut may soon die out.

Cytotoxic and DNA-topoisomerase effects of lapachol amine derivatives and interactions with DNA

The cytotoxic activity of amino (3a-e), aza-1-antraquinone (4a-e) lapachol derivatives against Ehrlich carcinoma and human K562 leukemia cells was investigated. Cell viability was determined using MTT assay, after 48 (Ehrlich) or 96 h (K562) of culture, and vincristine (for K562 leukemia) and quercetin (for Ehrlich carcinoma) were used as positive controls. The results showed dose-dependent growth-inhibiting activities and that the amino derivatives were active against the assayed cells, whereas the 4a-e derivatives were not. The allylamine derivative 3a was the most active against Ehrlich carcinoma, with IC50 = 16.94 ± 1.25 µM, and against K562 leukemia, with IC50 = 14.11 ± 1.39 µM. The analogous lawsone derivative, 5a, was also active against Ehrlich carcinoma (IC50 = 23.89 ± 2.3 µM), although the 5d and 5e derivatives showed lower activity. The interaction between 3a-d and calf thymus DNA was investigated by fluorimetric titration and the results showed a hyperchromic effect indicating binding to DNA as presented of ethidium bromide, used as positive control. The inhibitory action on DNA-topoisomerase II-a was also evaluated by a relaxation assay of supercoiled DNA plasmid, and the etoposide (200 µM) was used as positive control. Significant inhibitory activities were observed for 3a-d at 200 µM and a partial inhibitory action was observed for lapachol and methoxylapachol.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

In vitro studies of multiwalled carbon nanotube/ultrahigh molecular weight polyethylene nanocomposites with osteoblast-like MG63 cells

Carbon nanotubes are highly versatile materials; new applications using them are continuously being developed. Special attention is being dedicated to the possible use of multiwalled carbon nanotubes in biomaterials contacting with bone. However, carbon nanotubes are also controversial in regards to effects exerted on living organisms. Carbon nanotubes can be used to improve the tribological properties of polymer/composite materials. Ultrahigh molecular weight polyethylene (UHMWPE) is a polymer widely used in orthopedic applications that imply wear and particle generation. We describe here the response of human osteoblast-like MG63 cells after 6 days of culture in contact with artificially generated particles from both UHMWPE polymer and multiwalled carbon nanotubes (MWCNT)/UHMWPE nanocomposites. This novel composite has superior wear behavior, having thus the potential to reduce the number of revision hip arthroplasty surgeries required by wear failure of acetabular cups and diminish particle-induced osteolysis. The results of an in vitro study of viability and proliferation and interleukin-6 (IL-6) production suggest good cytocompatibility, similar to that of conventional UHMWPE (WST-1 assay results are reported as percentage of control ± SD: UHMWPE = 96.19 ± 7.92, MWCNT/UHMWPE = 97.92 ± 8.29%; total protein: control = 139.73 ± 10.78, UHMWPE = 137.07 ± 6.17, MWCNT/UHMWPE = 163.29 ± 11.81 µg/mL; IL-6: control = 90.93 ± 10.30, UHMWPE = 92.52 ± 11.02, MWCNT/UHMWPE = 108.99 ± 9.90 pg/mL). Standard cell culture conditions were considered as control. These results, especially the absence of significant elevation in the osteolysis inductor IL-6 values, reinforce the potential of this superior wear-resistant composite for future orthopedic applications, when compared to traditional UHMWPE.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15% of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80% sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

Modulation of murine blastocyst hatching in vitro by glutamine and tryptophan

Enrichment of culture media with amino acids improves embryo development. However, little is known about the specific action of each amino acid during embryogenesis. The present study was undertaken to examine the effect of L-glutamine (Gln) and tryptophan (Trp) on mouse embryo hatching, expansion and viability in vitro. Blastocysts were collected from 6- to 8-week-old female BALB/c mice (N = 30) and cultured in M2 medium containing either 0.125, 0.25 or 0.5 mM Trp, 1 mM Gln, or M2 alone. Gln significantly increased (100%; P < 0.05) blastocyst hatching at 24 h compared to M2 alone or Trp; moreover, Trp inhibited blastocyst hatching when compared to M2 alone (P < 0.05) at 72 h. In contrast, the percentage of embryos reaching the state of expanded blastocyst at 48 h was significantly higher in medium with 1 mM Gln (66.6%; P < 0.05) or with 0.125 mM Trp (61.1%; P < 0.05). Unexpectedly, Trp increased the percentage of degenerated blastocysts after 48 h (67.7%; P < 0.05), while Gln preserved blastocyst viability. These results suggest that Gln may enhance blastocyst hatching, expansion and viability in vitro.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles culturedin vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Affektitehdas. Työn rationalisoinnin historiallisia jatkumoita

Affektit, kapitalismi ja työn rationalisointi yhdistyvät toisiinsa erottamattomasti. Nykykeskusteluissa työstä ja työelämästä rationalisointi jaetaan usein tekniseen fordistiseen rationalisointiin ja sitä tunteellisempaan postfordistiseen rationalisointiin. Tämä tutkimus osoittaa, affektiivisen rationalisoinnin käsitteen kautta, että työn vanhat ja uudet tieteet asettuvat jatkumoon. Työn tieteissä tietoa työläisestä tuotetaan asettamalla ominaisuuksia aina uudelleen määriteltävään työläisen luokittuneeseen ja sukupuolittuneeseen affektiiviseen figuuriin. Tiedon tuottamisen näkökulmasta muun muassa tayloristiset työn tehostamisen fantasiat, ihmissuhdekoulukunnan ideologiat sekä uuden työn autonomiset järjestykset sisältävät yhtenäisiä piirteitä. Tutkimuksen pääkysymys on: miten affekteja rationalisoidaan työssä, työn tieteissä ja niiden kerrostumissa. Tutkimuksessa kysytään myös, miten affekti kiinnittää luokittuneet ja sukupuolittuneet subjektit työn rationalisoinnin aatteeseen ja millaisia mahdollisuuksia työläiseksi tulemiseen niissä tarjotaan. Kysymystä lähestytään monipuolisen aineiston ja lukuisten teemojen kautta. Aineistoina toimivat muun muassa rationalisoinnin klassikko-teokset, operaismo-vaikutteisen prekariaattiliikkeen pamfletit, ajatushautomoiden uuden työn visiot, self-help-teokset uranaisille sekä Helsingin Sanomien työelämäkeskustelut. Tutkimuksen monipuolista aineistoa lähiluetaan ja tulkitaan siihen luodun erityisen metodologian kautta, joka koostuu kolmesta osa-alueesta: historiallistaminen, paikantuminen ja figuurit. Kaksi ensimmäistä korostavat kontekstien ja position merkitystä tiedon tuotannossa, kolmas osoittaa tiedon tuottamisen valtasuhteita. Työn tieteiden visiot ideaalityöläisestä materialisoituvat figuurien kautta negaatioina kuvaten sitä, millainen ideaalityöläinen ei ole. Väitöskirja aineistoineen paikantuu Suomeen, mutta se osoittaa työn tieteiden ja affektiivisen rationalisoinnin globaaliutta sekä sidonnaisuutta Yhdysvaltoihin, sen yrityskulttuuriin ja esimerkiksi työtehotutkimuksiin Hawthornen elektroniikkatehtaalla vuosina 1924–1933. Väitöskirja tuo uusia näkökulmia nykykeskusteluun työstä, työläisistä, kapitalismista ja affekteista. Se osoittaa, että itsen tuntemisen ja kertomisen tieteitä, kuten psykologiaa, tarjotaan usein ratkaisuksi kapitalistisen tuotannon aiheuttamiin suuriin ja pieniin kurjuuksiin, vaikka terapeuttinen tunnekulttuuri itsessään on muodostunut osana rationalisoinnin ja kapitalismin ambivalentteja kehiä. Tutkimus myös muistuttaa, että työn järjestyksien keskiössä säilyy työläisen ja affektitehtaan konflikti silloinkin, kun fordistinen imperatiivi ”älä!” vaihtuu postfordistiseen kehotukseen ”tunne”.