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Samik Basu,Tatiana Golovina,Tatiana Mikheeva,Carl H June,James L Riley Addition of rapamycin to cultures of expanding natural CD4+CD25+Foxp3+ T regulatory cells (Tregs) helps maintain their suppressive activity, but the underlying mechanism is unclear. Pim 2 is a serine/threonine kinase that can confer rapamycin resistance. Unexpectedly, pim 2 was found to be constitu... ( view more )tively expressed in freshly isolated, resting Tregs, but not in CD4+CD25- T effector cells. Introduction of Foxp3, but not Foxp3Delta2, into effector T cells induced pim 2 expression and conferred preferential expansion in the presence of rapamycin, indicating that Foxp3 can regulate pim 2 expression. Finally, we determined there is a positive correlation between Treg expansion and Foxp3 expression in the presence of rapamycin. Together, these results indicate that Tregs are programmed to be resistant to rapamycin, providing further rationale for why this immunosuppressive drug should be used in conjunction with expanded Tregs. ( view less ) Valerij Ya Grinberg,Tatiana V Burova,Natalia V Grinberg,Tatiana A Shcherbakova,Dorel T Guranda,Ghermes G Chilov,Vytas K Svedas Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 ... ( view more )to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed. ( view less ) Rong-Jian Lu,John A Tucker,Tatiana Zinevitch,Olga Kirichenko,Vitalii Konoplev,Svetlana Kuznetsova,Sergey Sviridov,Jason Pickens,Sagun Tandel,Enugurthi Brahmachary,Yang Yang,Jian Wang,Stephanie Freel,Shelly Fisher,Alana Sullivan,Jiying Zhou,Sherry Stanfield-Oakley,Michael Greenberg,Dani Bolognesi,Brian Bray,Barney Koszalka,Peter Jeffs,Alisher Khasanov,You-An Ma,Cynthia Jeffries,Changhui Liu,Tatiana Proskurina,Tong Zhu,Alexander Chucholowski,Rongshi Li,Connie Sexton The crystal structures of many tertiary alpha-ketoamides reveal an orthogonal arrangement of the two carbonyl groups. Based on the hypothesis that the alpha-ketoamide HIV attachment inhibitor BMS 806 (formally BMS378806, 26) might bind to its gp120 target via a similar conformation, we designed and... ( view more ) synthesized a series of analogs in which the ketoamide group is replaced by an isosteric sulfonamide group. The most potent of these analogs, 14i, demonstrated antiviral potency comparable to 26 in the M33 pseudotyped antiviral assay. Flexible overlay calculations of a ketoamide inhibitor with a sulfonamide inhibitor revealed a single conformation of each that gave significantly better overlap of key pharmacophore features than other conformations and thus suggest a possible binding conformation for each class. ( view less ) Daniela Campagnol,Francisco J Teixeira Neto,Tatiana Giordano,Tatiana H Ferreira,Eduardo R Monteiro Objective-To evaluate the effects of epidural administration of 3 doses of dexmedetomidine on isoflurane minimum alveolar concentration (MAC) and characterize changes in bispectral index (BIS) induced by nociceptive stimulation used for MAC determination in dogs. Animals-6 adult dogs. Procedures-Is... ( view more )oflurane-anesthetized dogs received physiologic saline (0.9% NaCl) solution (control treatment) or dexmedetomidine (1.5 [DEX1.5], 3.0 [DEX3], or 6.0 [DEX6] mug/kg) epidurally in a crossover study. Isoflurane MAC (determined by use of electrical nociceptive stimulation of the hind limb) was targeted to be accomplished at 2 and 4.5 hours. Changes in BIS attributable to nociceptive stimulation and cardiopulmonary data were recorded at each MAC determination. Results-With the control treatment, mean +/- SD MAC values did not change over time (1.57 +/- 0.23% and 1.55 +/- 0.25% at 2 and 4.5 hours, respectively). Compared with the control treatment, MAC was significantly lower at 2 hours (13% reduction) but not at 4.5 hours (7% reduction) in DEX1.5-treated dogs and significantly lower at 2 hours (29% reduction) and 4.5 hours (13% reduction) in DEX3-treated dogs. The DEX6 treatment yielded the greatest MAC reduction (31% and 22% at 2 and 4.5 hours, respectively). During all treatments, noxious stimulation increased BIS; but changes in BIS were correlated with increases in electromyographic activity. Conclusions and Clinical Relevance-In dogs, epidural administration of dexmedetomidine resulted in dose-dependent decreases in isoflurane MAC and that effect decreased over time. Changes in BIS during MAC determinations may not represent increased awareness because of the possible interference of electromyographic activity. ( view less ) Raquel Guimarães-Costa,Maria Beatriz Guimarães-Costa,Leonardo Pippa-Gadioli,Alfredo Weltson,Walter Adriano Ubiali,Tatiana Paschoalin-Maurin,Tatiana Tocchini Felippotti,Daoud Hibrahim Elias-Filho,Carlos Júlio Laure,Norberto Cysne Coimbra Defensive behaviour has been extensively studied, and non-invasive methodologies may be interesting approaches to analyzing the limbic system function as a whole. Using experimental models of animals in the state of anxiety has been fundamental in the search for new anxiolytic and antipanic compoun... ( view more )ds. The aim of this present work is to examine a new model for the study of affective behaviour, using a complex labyrinth consisting of an arena and galleries forming a maze. Furthermore, it aims to compare the defensive behaviour of Wistar rats, Mongolian gerbils and golden hamsters in a complex labyrinth, as well as the defensive behaviour of Meriones unguiculatus in aggressive encounters with either Epicrates cenchria assisi or Boa constrictor amarali in this same model. Among species presently studied, the Mongolian gerbils showed better performance in the exploration of both arena and galleries of the labyrinth, also demonstrating less latency in finding exits of the galleries. This increases the possibility of survival, as well as optimizes the events of encounter with the predator. The duration of alertness and freezing increased during confrontation with living Epicrates, as well as the duration of exploratory behaviour in the labyrinth. There was an increase in the number of freezing and alertness behaviours, as well as in duration of alertness during confrontations involving E.c. assisi, compared with behavioural reactions elicited by jirds in presence of B.c. amarali. Interestingly, the aggressive behaviour of Mongolian gerbils was more prominent against B.c. amarali compared with the other Boidae snake. E.c. assisi elicited more offensive attacks and exhibited a greater time period of body movement than B.c. amarali, which spent more time in the arena and in defensive immobility than the E.c. assisi. Considering that jirds evoked more fear-like reaction in contact with E.c. assisi, a fixed E.c. assisi kept in a hermetically closed acrylic box was used as control. In these prey/predator encounter-based experiments, there was an increase in the number of alertness and freezing behaviours exhibited by gerbils, and a decrease in the number of crossing elicited by them, when comparing confrontations between the living E.c. assisi and the control. The experiments were performed at 7.0 p.m. In the labyrinth, the snakes showed in confrontation similar performance to that observed in nature (organizing hunting behaviour, offensive/defensive attack, constriction, prey inspection and feeding behaviour), which were essential to the validity of the experiments and gave behavioural validation within the complex labyrinth. ( view less ) Ludmila M Khromykh,Natalia L Kulikova,Tatiana V Anfalova,Tatiana A Muranova,Vyacheslav M Abramov,Anatoliy M Vasiliev,Valentin S Khlebnikov,Dmitriy B KazanskySupernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine ... ( view more )cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells. ( view less ) Stacy Zamudio,Lucrecia Postigo,Nicholas P Illsley,Carmelo Rodriguez,Gladys Heredia,Michael Brimacombe,Lourdes Echalar,Tatiana Torricos,Wilma Tellez,Ivan Maldonado,Elfride Balanza,Tatiana Alvarez,Julio Ameller,Enrique Vargas Fetal growth is reduced at high altitude, but the decrease is less among long-resident populations. We hypothesized that greater maternal uteroplacental O(2) delivery would explain increased fetal growth in Andean natives versus European migrants to high altitude. O(2) delivery was measured with ul... ( view more )trasound, Doppler and haematological techniques. Participants (n=180) were pregnant women of self-professed European or Andean ancestry living at 3600 m or 400 m in Bolivia. Ancestry was quantified using ancestry-informative single nucleotide polymorphism. The altitude-associated decrement in birth weight was 418 g in European versus 236 g in Andean women (P<0.005). Altitude was associated with decreased uterine artery diameter, volumetric blood flow and O(2) delivery regardless of ancestry. But the hypothesis was rejected as O(2) delivery was similar between ancestry groups at their respective altitudes of residence. Instead, Andean neonates were larger and heavier per unit of O(2) delivery, regardless of altitude (P<0.001). European admixture among Andeans was negatively correlated with birth weight at both altitudes (P<0.01), but admixture was not related to any of the O(2) transport variables. Genetically mediated differences in maternal O(2) delivery are thus unlikely to explain the Andean advantage in fetal growth. Of the other independent variables, only placental weight and gestational age explained significant variation in birth weight. Thus greater placental efficiency in O(2) and nutrient transport, and/or greater fetal efficiency in substrate utilization may contribute to ancestry- and altitude-related differences in fetal growth. Uterine artery O(2) delivery in these pregnancies was 99 +/- 3 ml min(-1), approximately 5-fold greater than near-term fetal O(2) consumption. Deficits in maternal O(2) transport in third trimester normal pregnancy are unlikely to be causally associated with variation in fetal growth. ( view less ) Ludmila Filaretova,Tatiana Podvigina,Tatiana Bagaeva,Peter Bobryshev,Koji Takeuchi Gastric ulcer disease remains widespread; a stressful lifestyle and nonsteroidal antiinflammatory drugs (NSAIDs) make significant contributions to this pathological situation. The findings overviewed here support the idea that glucocorticoid hormones released in response to acute stress or NSAIDs a... ( view more )ct as gastroprotective substances and exert many of the same actions in the stomach as prostaglandins (PGs) and nitric oxide (NO) as well as capsaicin-sensitive afferent neurons. Glucocorticoids exert a gastroprotective effect by both maintaining local defensive factors (mucosal blood flow and mucus production) and inhibiting pathogenic elements (gastric motility and microvascular permeability). Furthermore, they exert gastroprotective actions in co-operation with PGs, NO, and the afferent neurons; and their compensatory action is observed when the protective mechanism provided by either of these factors is impaired. The gastroprotective action of glucocorticoids is also associated with maintenance of general body homeostasis, including blood glucose levels and systemic blood pressure. In conclusion, glucocorticoids released in response to acute stress or NSAIDs are naturally occurring protective factors that play an important role in maintenance of the gastric mucosal integrity. This led us to re-evaluate the traditional paradigm that glucocorticoid hormones produced during activation of the hypothalamic-pituitary-adrenocortical axis are ulcerogenic in the stomach. ( view less ) Tatiana Indelicato da Silva,Rozana Mesquita Ciconelli,Neide Barreira Alonso,Auro Mauro Azevedo,Ana Carolina Westphal-Guitti,Tatiana Frascarelli Pascalicchio,Carolina Mattos Marques,Luís Otávio Sales Ferreira Caboclo,Joyce A Cramer,Américo Ceiki Sakamoto,Elza Márcia Targas Yacubian PURPOSE: We report the cultural adaptation and psychometric properties of the Quality of Life in Epilepsy-31 Inventory (QOLIE-31) for the Portuguese language and Brazilian culture. METHODS: This study involved 150 outpatients: 50 presurgical patients with refractory temporal lobe epilepsy (TLE) rel... ( view more )ated to mesial temporal sclerosis (MTS), 50 patients with juvenile myoclonic epilepsy (JME), and 50 seizure-free patients with TLE. They completed the QOLIE-31, Nottingham Health Profile (NHP), Beck Depression Inventory (BDI), and Adverse Events Profile (AEP) and underwent a neuropsychological evaluation (NE). Internal consistency reliability, interrater and test-retest reliability, and construct validity were assessed. RESULTS: QOLIE-31 mean scores were 33.1 (Social Function), 68.9 (Overall Quality of Life), 56.5 (Seizure Worry), 64.1 (Emotional Well-Being), 63.7 (Energy/Fatigue), 38.9 (Cognitive Function), and 49.7 (Medication Effects). Internal consistency was high (Cronbach's alpha), as were the associations between QOLIE-31 and the BDI, NHP, AEP, and NE. CONCLUSION: The Portuguese/Brazilian version of the QOLIE-31 inventory showed good reliability, validity, and construct validity. ( view less ) Michail Shipitsin,Lauren L Campbell,Pedram Argani,Stanislawa Weremowicz,Noga Bloushtain-Qimron,Jun Yao,Tatiana Nikolskaya,Tatiana Serebryiskaya,Rameen Beroukhim,Min Hu,Marc K Halushka,Saraswati Sukumar,Leroy M Parker,Karen S Anderson,Lyndsay N Harris,Judy E Garber,Andrea L Richardson,Stuart J Schnitt,Yuri Nikolsky,Rebecca S Gelman,Kornelia Polyak Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells pu... ( view more )rified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations. ( view less ) Stanislav A Geras'kin,Tatiana I Evseeva,Elena S Belykh,Tatiana A Majstrenko,Boguslaw Michalik,Anatoliy I Taskaev Results of long-term radioecological investigations in areas with an enhanced level of natural radioactivity in the north of Russia are summarized. Deleterious changes within animal and plant populations inhabiting areas with an enhanced level of natural radioactivity in the Komi Republic were reve... ( view more )aled. These changes are expressed in enhanced levels of mutagenesis, destructive processes in the tissues of animals, disturbances of reproductive functions and reduced offspring viability. Compensatory processes, resulting in animal and plant survival under extremely adverse conditions of radium and uranium-radium contamination, were observed as well. However, obvious signs of adaptation failed to be detected. The findings suggest that adverse somatic and genetic effects are possible in plants and animals in the dose range observed at sites with an enhanced level of natural radioactivity. In contrast, different plant species inhabiting an area with an enhanced level of natural radioactivity in the taiga zone of the Sakha Republic (Yakutia) reveal a stimulation of growth processes, photosynthesis, endogenous low molecular weight antioxidant synthesis as well as adaptive response. It is apparent from the data presented that naturally occurring differences in terrestrial radiation level could be of genetic and ecological consequence. ( view less ) Marcelo Schmitz,Daniel Denardin,Tatiana Laufer Silva,Thiago Pianca,Tatiana Roman,Mara Helena Hutz,Stephen V Faraone,Luis Augusto Rohde BACKGROUND: Previous investigations have demonstrated that an MspI polymorphism at the adrenergic alpha2A receptor gene (ADRA2A) is associated with severity of attention-deficit/hyperactivity disorder (ADHD) inattentive symptoms in clinical samples composed mainly of subjects with ADHD, combined ty... ( view more )pe. This study aimed to investigate the association between this ADRA2A polymorphism and attention-deficit/hyperactivity disorder-inattentive type (ADHD-I) in a nonreferred sample. METHODS: In a case-control study, we assessed a sample of 100 children and adolescents with ADHD-I and 100 non-ADHD controls. Cases and controls were matched by gender and age and were screened by using teacher reports in a revised version of the Swanson, Nolan, and Pelham rating scale at 12 schools. Psychiatric diagnoses were derived through structured diagnostic interviews. RESULTS: Homozygous subjects for the G allele at the ADRA2A had significantly higher odds ratio (OR) for ADHD-I than did those with other genotypes (CC + CG genotypes), even after adjusting for potential confounders (p = .02; OR = 3.78; 95% confidence interval = 1.23-11.62). In family-based analyses, no significant associations were detected. CONCLUSIONS: Our results suggest that the ADRA2A may be associated with ADHD-I, replicating previous findings from clinical samples that have suggested the importance of this gene for the dimension of inattention. In addition, these results support the role of the noradrenergic system in ADHD. ( view less ) Elena E Petrova,Tatiana I Valyakina,Maria A Simonova,Ravilya L Komaleva,Sergey V Khaidukov,Eugene A Makarov,Dmitry Yu Blokhin,Pavel K Ivanov,Tatiana M Andronova,Vladimir A Nesmeyanov We have demonstrated that biologically active muramyl peptides, in particular, glucosaminylmuramyl dipeptide (GMDP), augmented in vitro cytotoxic activity of tumor necrosis factor-alpha (TNF-alpha) against murine fibrosarcoma L929 cells. The introduction of GMDP resulted in cytotoxic effect charact... ( view more )eristic for substantially higher dose of cytokine. Even more potent was the combination of GMDP, TNF-alpha and Actinomycin D (ActD). According to clonogenic and MTT assays 100% L929 cells could be killed in culture with low doses of TNF-alpha and ActD if GMDP was present. When cisplatin was substituted for ActD similar results were obtained. GMDP also enhanced cytotoxicity of TNF-alpha and cisplatin against human breast carcinoma MCF7 and histiocytic lymphoma U937 cells. Normal cells, namely human peripheral blood leucocytes and murine peritoneal macrophages, were resistant to selected doses of TNF-alpha/cisplatin/GMDP. ( view less ) Tatiana D Sirakova,Vinod S Dubey,Chirajyoti Deb,Jaiyanth Daniel,Tatiana A Korotkova,Bassam Abomoelak,Pappachan E Kolattukudy Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv... ( view more ) accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C(26) was a major fatty acid in this TG, indicating that the TGS1 gene product uses C(26) fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C(26 : 0)-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs. ( view less ) Sergei N Shchelkunov,Rurik K Salyaev,Sergei G Pozdnyakov,Natalia I Rekoslavskaya,Andrei E Nesterov,Tatiana S Ryzhova,Valentina M Sumtsova,Natalia V Pakova,Uliana O Mishutina,Tatiana V Kopytina,Rosemarie W HammondA synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice an... ( view more )d, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV. ( view less ) Tatiana N Drebushchak,Natalia N Pankrushina,Tatiana P Shakhtshneider,Svetlana A ApeninaThe crystal structure of the title compound, C(22)H(17)N(3)O(5)S, contains dimers linked by N-H...O hydrogen bonds about inversion centers. The dimers are packed in a herring-bone framework without classical hydrogen bonds between the structure-forming units. Tatiana I Rogounovitch,Vladimir A Saenko,Kiyoto Ashizawa,Ilya A Sedliarou,Hiroyuki Namba,Aleksandr Yu Abrosimov,Eugeny F Lushnikov,Pavel O Roumiantsev,Marina V Konova,Natalia S Petoukhova,Irina V Tchebotareva,Viktor K Ivanov,Sergei Yu Chekin,Tatiana I Bogdanova,Mykola D Tronko,Anatoly F Tsyb,Gerry A Thomas,Shunichi Yamashita The study investigated an association between the germline polymorphism at TP53 codon 72 and the development of papillary thyroid cancer (PTC) following exposure to radiation from the Chernobyl accident. TP53 genotype was examined in 48 pediatric/adolescent (age at diagnosis <18 years) and 68 adult... ( view more ) post-Chernobyl patient with PTC, 53 adult patients with sporadic PTC and 313 healthy individuals from Russian-Ukrainian population. In addition, we evaluated loss of heterozygosity for TP53 and the allele expression ratio. The genotype of the patients was correlated with clinicopathological data. Arg TP53 homozygotes were found to be significantly underrepresented among adults with post-Chernobyl PTC, but not in children and adolescents when compared with sporadic PTC cases and the general population. In the tumors, cell transformation did not lead to allelic loss or biased TP53 allele expression in heterozygous individuals. None of TP53 genotypes specifically associated with tumor stage and morphology, however there were particular correlations with lymph node status in certain age groups of radiation-associated cases not seen in sporadic PTCs. The findings suggest TP53 allele combinations other than Arg/Arg may contribute to the risk of development of PTC in individuals exposed to radiation during their late childhood, adolescence or in young adulthood. ( view less ) Tatiana I Rosca,Monica I Pop,Manuela Curca,Teodora Gh Vladescu,Cecilia St Tihoan,Ana Tatiana T Serban,Ecaterina Al Bontas,Gheorghe GherghescuVascular tumors in the orbit result from new formation of vessels, proliferation of tissue components of the vessel wall, and hyperplasia of cellular elements ordinarily concerned with the genesis of vascular tissue. These vasculogenic lesions constitute the largest group of primary orbital tumors;... ( view more ) we present the capillary hemangioma and the cavernous hemangioma. ( view less ) Tatiana V Kondrashova,Kazuo Neriishi,Sadayuki Ban,Tatiana I Ivanova,Lyudmila I Krikunova,Nataliya I Shentereva,Iya A Smirnova,Irina A Zharikova,Marina V Konova,Senjun Taira,Anatoly F Tsyb Possible association between the C282Y and H63D mutations in the HFE gene and estrogen-dependent cancer risk was assessed. Genotyping was performed using PCR amplification followed by digestion of products with specific restrictases. In a population of 260 healthy women (permanent residents of the ... ( view more )southwest European Russia), mutant allele frequencies at the C282Y and H63D sites were evaluated as 3.3 and 16.3%, respectively. In patients with breast, ovarian, and endometrial cancer, C282Y frequencies were also low (1.0, 1.3, and 3.8%, respectively), and no cancer risk associated with the C282Y mutation was found. Odds ratios for breast cancer risk associated with the H63D mutation increased significantly with age: 0.5 in women below 48 years old, 1.0 in a range of 48-57 years, and 4.4 in older women (P(trend)=0.002). The latter value was statistically significant (95% CI, 1.4-14.1), indicating that women bearing the H63D mutation may be at an increased breast cancer risk at an age above 57 years. Preliminary results obtained in patients with two other estrogen-dependent malignancies revealed the same tendency to OR increase with age in ovarian cancer patients (P(trend)=0.008), but no age-related OR differences in endometrial cancer patients. ( view less ) Tatiana Shaposhnikova,Ivan Matveev,Tatiana Napara,Olga Podgornaya The extracellular matrix of the jellyfish Aurelia aurita (Scyphozoa, Cnidaria), known as the mesoglea, is populated by numerous mesogleal cells (Mc). We determined the pattern of the Mc and the mesoglea, raised polyclonal antibodies (RA47) against the major mesogleal protein pA47 (47 kDa) and check... ( view more )ed their specificity. In the mesoglea, RA47 stains pA47 itself. In immunoblots of Mc, RA47 stains bands of 120 kDa and 80 kDa; weaker staining is observed at pA47. The same staining pattern is seen on blots of jellyfish epidermal cells and of whole Hydra (Hydrozoa) or isolated mesoglea of Hydra. Our data indicate that pA47 is synthesized by Mc and epidermal cells as high molecular precursors. Using immunostaining techniques, we showed Mc to be involved in the formation of mesogleal non-collagenous (called "elastic" in classic morphological studies) fibres. The biochemical and morphological data suggest that Mc originate from the epidermis. ( view less ) Tatiana I Arefieva,Nadezhda B Kukhtina,Olga A Antonova,Tatiana L Krasnikova Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understo... ( view more )od. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity. ( view less ) Valérie Heurgué-Hamard,Stéphanie Champ,Liliana Mora,Tatiana Merkulova-Rainon,Tatiana Merkoulova-Rainon,Lev L Kisselev,Richard H Buckingham Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is... ( view more ) modified to N(5)-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N(5)-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1.eRF3.GTP. ( view less ) Sergei Degtyarev,Tatiana Boykova,Andrei Grishanin,Stepan Belyakin,Nikolai Rubtsov,Tatiana Karamysheva,Grigory Makarevich,Alexei Akifyev,Igor Zhimulev Presumptive somatic cells of the copepod Cyclops kolensis specifically eliminate a large fraction of their genome by the process of chromatin diminution. The eliminated DNA (eDNA) remains only in the germline cells. Very little is known about the nature of the sequences eliminated from somatic cell... ( view more )s. We cloned a fraction of the eDNA and sequenced 90 clones that total 32 kb. The following organizational patterns were demonstrated for the eDNA sequences. All do not contain open reading frames. Each fragment contains 1-3 families of short repeats (10-30 bp) highly homologous within families (87%-100%). Most repeats are separated by spacers up to 50 bp long. Homologous regions were found between fragments, motifs from 15-300 bp in length. Among fragments there occur groups in which the same motifs are ordered in the same fashion. However, spacers between the motifs differ in length and nucleotide composition. Ubiquitous motifs (those occurring in all fragments) were identified. Analysis of motifs revealed submotifs, each occurring within several motifs. Thus, motifs may be regarded as mosaic structures composed of submotifs (short repeats). Taken together, the results provide evidence of a high organizational ordering of the DNA sequences restricted to the germline. With this in mind, it appears incorrect to refer to this part of the genome as junk. Moreover, eDNA is redundant for only the somatic cells-its function is to be sought in germline cells. ( view less ) Tatiana Mareeva,Tatiana Lebedeva,Nadia Anikeeva,Tim Manser,Yuri Sykulev Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of ... ( view more )the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo. ( view less ) Elisabete Freire,Flávia C A Gomes,Tatiana Jotha-Mattos,Vivaldo Moura Neto,Fernando C Silva Filho,Tatiana Coelho-Sampaio In the developing nervous system migrating neurons and growing axons are guided by diffusible and/or substrate-bound cues, such as extracellular matrix-associated laminin. In a previous work we demonstrated that laminin molecules could self-assemble in two different manners, giving rise to matrices... ( view more ) that could favor either neuritogenesis or proliferation of cortical precursor cells. We investigated whether the ability of astrocytes to promote neuritogenesis of co-cultivated neurons was modulated by the assembling mode of the laminin matrix secreted by them. We compared the morphologies and neuritogenic potentials of laminin deposited by in vitro-differentiated astrocytes obtained from embryonic or neonatal rat brain cortices. We showed that, while permissive astrocytes derived from embryonic brain produced a flat laminin matrix that remained associated to the cell surface, astrocytes derived from newborn brain secreted a laminin matrix resembling a fibrillar web that protruded from the cell plane. The average neurite lengths obtained for E16 neurons cultured on each astrocyte layer were 198+/-22 and 123+/-13 microm, respectively. Analyses of surface-associated electrostatic potentials revealed that embryonic astrocytes presented a pI of -2.8, while in newborn cells this value was -3.8. Removal of the sialic acid groups on the embryonic monolayer by neuraminidase treatment led to the immediate release of matrix-associated laminin. Interestingly, laminin reassembled 1 hour after neuraminidase removal converted to the features of the newborn matrix. Alternatively, treatment of astrocytes with the cholesterol-solubilizing detergent methyl-beta-cyclodextrin also resulted in release of the extracellular laminin. To test the hypothesis that sialic-acid-containing lipids localized at cholesterol-rich membrane domains could affect the process of laminin assembly, we devised a cell-free assay where laminin polymerization was carried out over artificial lipid films. Films of either a mixture of gangliosides or pure ganglioside GT1b induced formation of matrices of morpho-functional features similar to the matrices deposited by embryonic astrocytes. Conversely, films of phosphatidylcholine or ganglioside GM1 led to the formation of bulky laminin aggregates that lacked a defined structure. We propose that the expression of negative lipids on astrocytes can control the extracellular polymerization of laminin and, consequently, the permissivity to neuritogenesis of astrocytes during development. ( view less )
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