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Maturation Process (maturation + process)
Selected AbstractsTheoretical Study of Catalytic Efficiency of a Diels,Alderase Catalytic Antibody: An Indirect Effect Produced During the Maturation ProcessCHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008Sergio Martí Dr. Abstract The Diels,Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39,A-11, that catalyze a Diels,Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate,protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate. [source] Gazing at the Hand: A Foucaultian View of the Teaching of Manipulative Skills to Introductory Chemistry Students in the United States and the Potential for Transforming Laboratory InstructionCURRICULUM INQUIRY, Issue 3 2005STEPHEN DEMEO ABSTRACT Many studies of chemistry have described the rise of the academic chemical laboratory and laboratory skills in the United States as a result of famous men, important discoveries, and international influences. What is lacking is a perspective of the manifestations of the balances of power and knowledge between teacher and student. A Foucaultian analysis of the teaching of manipulative skills to the introductory student in high school and college in the United States during the later half of the 19th and into the 20th century has provided such a perspective. The analysis focuses on the body, specifically students' hands, and how this body has been redescribed in terms of time, space, activity, and their combinations. It is argued in the first part of this article that the teaching of manipulative skills in the chemistry laboratory can be characterized by effects of differential forms of power and knowledge, such as those provided by Foucault's ideas of hierarchical observation, normalization, and the examination. Moreover, it is evident that disciplinary techniques primarily focused on the physical hands of the student have been recast to include a new cognitive-physiological space in which the teaching of manipulative skills currently takes place. In the second part of this article, the author describes his own professional development as a laboratory instructor through a series of reflective statements that are critiqued from a Foucaultian perspective. The personal narratives are presented in order to pro- vide science educators with an alternative way for their students to think about the relationship between one's manipulative skills and the quality of their data. The pedagogical approach is related to the maturation process of the chemist and contextualized in the current paradigm of laboratory practice, inquiry-based science education. [source] Traumatic injuries to the primary dentition and effects on the permanent successors , a clinical follow-up studyDENTAL TRAUMATOLOGY, Issue 5 2006Sabine Sennhenn-Kirchner Abstract,,, This study investigated problems in the permanent dentition that, according to history and records, were attributable to dental alveolar injuries of the primary dentition. 106 children have been involved in the study, who had experienced primary anterior tooth trauma affecting a total of 200 teeth. Thirty-nine patients (81 teeth) were available for follow-up examinations. In 25% of the cases followed up, damage was found on the successors in the secondary dentition (16 children/20 teeth). In half of the cases, a comparatively mild form of lesion like enamel discoloration was observed. This was the result of an injury during the tooth maturation process causing enamel hypoplasia. Clinically more relevant were the dental deformities: cessation of root formation or retention caused by ankylosis, which made up the remaining 50% of cases. This was confirmed by clinical long-term observation. The different effects on the permanent teeth can only be detected by radiography after an interval of several months or may even be clinically assessed only after the eruption of the clinical crown. [source] MEASURING PROBABILISTIC REACTION NORMS FOR AGE AND SIZE AT MATURATIONEVOLUTION, Issue 4 2002Mikko Heino Abstract We present a new probabilistic concept of reaction norms for age and size at maturation that is applicable when observations are carried out at discrete time intervals. This approach can also be used to estimate reaction norms for age and size at metamorphosis or at other ontogenetic transitions. Such estimations are critical for understanding phenotypic plasticity and life-history changes in variable environments, assessing genetic changes in the presence of phenotypic plasticity, and calibrating size- and age-structured population models. We show that previous approaches to this problem, based on regressing size against age at maturation, give results that are systematically biased when compared to the probabilistic reaction norms. The bias can be substantial and is likely to lead to qualitatively incorrect conclusions; it is caused by failing to account for the probabilistic nature of the maturation process. We explain why, instead, robust estimations of maturation reaction norms should be based on logistic regression or on other statistical models that treat the probability of maturing as a dependent variable. We demonstrate the utility of our approach with two examples. First, the analysis of data generated for a known reaction norm highlights some crucial limitations of previous approaches. Second, application to the northeast arctic cod (Gadus morhua) illustrates how our approach can be used to shed new light on existing real-world data. [source] The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesisFEBS JOURNAL, Issue 5 2002Tomoko Kaneko Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage. [source] Differential expression of a Bombyx mori AHA1 homologue during spermatogenesisINSECT MOLECULAR BIOLOGY, Issue 3 2005Y. Miyagawa Abstract The AHA1 (activator of Hsp90 ATPase) family of proteins were exclusively conserved from yeast to humans, but little is known about their tissue distribution or biological function. In this study, a cDNA for a Bombyx mori AHA1 homologue, BmAHA1, was isolated from the testes of larvae on day 3 of the fifth instar using an mRNA differential display method. This cDNA encodes a protein with 341 amino acid residues. Gene expression studies revealed that BmAHA1 mRNA occurred prominently in the testes. In situ hybridization and immunostaining showed that the BmAHA1 mRNA signals were strongly detected in spermatogonial cells and primary spermatocytes at the fifth larval instar stage, whereas the BmAha1 protein was abundant in round and elongated spermatids at the pupal stage. The localization pattern of the accumulated protein in the elongated spermatids was reminiscent of that reported previously for microtubules, but the BmAha1 protein showed a decrease in apparent concentration during maturation process. The stage- and cell-specific expression indicated that BmAha1 might play a role in silkworm spermatogenesis, especially in postmeiotic differentiation. [source] Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx moriINSECT SCIENCE, Issue 1 2010Cheng-Long Wang Abstract, Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS. Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others. [source] Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response elementJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Arthur J. Cohen Abstract Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Expression of signal transduction proteins during the differentiation of primary human erythroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Viviana di Giacomo The high number (>108,10) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72,96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC ,4 was not expressed, PLC ,2, ,1, and ,2 were expressed at constant levels throughout the maturation process, while expression of PLC ,3 and of PLC ,1 decreased, as PI-3K, by 24 h and that of PLC ,1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC ,1, ,3, and ,1) might e involved in the control of erythroid differentiation in humans. © 2004 Wiley-Liss, Inc. [source] Presenilin 1 is involved in the maturation of ,-site amyloid precursor protein-cleaving enzyme 1 (BACE1)JOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007Akira Kuzuya Abstract One of the pathologic hallmarks of Alzheimer's disease is the excessive deposition of ,-amyloid peptides (A,) in senile plaques. A, is generated when ,-amyloid precursor protein (APP) is cleaved sequentially by ,-secretase, identified as ,-site APP-cleaving enzyme 1 (BACE1), and ,-secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of A, peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin-deficient mouse embryonic fibroblasts (PS,/,MEFs) were reduced significantly compared to those in wild-type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH-SY5Y, stably expressing wild-type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant,negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH-SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both ,- and ,-secretase in A, generation. © 2006 Wiley-Liss, Inc. [source] Detection of fascin and CCR-7 positive mature dendritic cells in oral lichen planusJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2009Shotaro Mukae Background:, Dendritic cells (DC) play a crucial role in the pathogenesis of oral lichen planus (OLP) with respect to antigens presented to T cells. We performed immunohistochemical analysis to elucidate the process of activation of DC in OLP. Methods:, Thirty biopsy specimens were obtained from the patients with OLP. The expressions of CD1a, Langerin, S-100, fascin, chemokine receptor-7 (CCR-7), D2-40, cyclooxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) in DC from OLP and disease free control were investigated using specific antibodies. The distribution and number (1 mm2) of DC were assessed in the intra-epithelium and the submucosa specimens. Correlation between the number of DC and epithelium thickness was also determined. Result:, Immature DC (Langerin+, CD1a+, and S-100+) were identified in the epithelia from OLP patients and control, though the numbers of Langerin+ and CD1a+ positive cells were decreased in the OLP samples as compared to the control. Mature DC (fascin+) were identified in the submucosa specimens, not found in the epithelium from OLP or control. Double immunostaining revealed DC positive for fascin and CCR-7 in the submucosa, which had migrated into D2-40+ lymph vessels. Furthermore, keratinocytes expressed both Prostaglandin E2 (PGE2) converting enzymes, COX-2, and mPGES-1, indicating PGE2 synthesis in the epithelial layer of the OLP specimens. Conclusion:, Our results indicate that DC change from immature to mature in the epithelium and are then drawn out to the submucosa. We demonstrate that mature DC localized in the submucosa, it consequently migrates into lymph vessels. This maturation process of DC is an important immunopathological feature of OLP. [source] Intervertebral disc cell response to dynamic compression is age and frequency dependent,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Casey L. Korecki Abstract The maintenance of the intervertebral disc extracellular matrix is regulated by mechanical loading, nutrition, and the accumulation of matrix proteins and cytokines that are affected by both aging and degeneration. Evidence suggests that cellular aging may lead to alterations in the quantity and quality of extracellular matrix produced. The aims of this study were to examine the role of loading and maturation (a subset of aging), and the interaction between these two factors in intervertebral disc cell gene expression and biosynthesis in a controlled 3D culture environment. Cells were isolated from young (4,6 months) and mature (18,24 months) bovine caudal annulus fibrosus and nucleus pulposus tissue. Isolated cells were seeded into alginate and dynamically compressed for 7 days at either 0.1, 1, or 3 Hz or maintained as a free-swelling control. After 7 days, DNA and sulfated glycosaminoglycan contents were analyzed along with real time, quantitative reverse transcription-polymerase chain reaction analysis for collagen types I and II, aggrecan, and matrix metalloproteinase-3 gene expression. Results suggest that maturation plays an important role in intervertebral disc homeostasis and influences the cell response to mechanical loading. While isolated intervertebral disc cells responded to mechanical compression in 3D culture, the effect of loading frequency was minimal. Altered cellular phenotype and biosynthesis rates appear to be an attribute of the cell maturation process, potentially independent of changes in cellular microenvironment associated with lost nutrition and disc degeneration. Mature cells may have a decreased capacity to create or retain extracellular matrix components in response to mechanical loading compared to young cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 800,806, 2009 [source] Structural motifs in the maturation process of peptide hormones.JOURNAL OF PEPTIDE SCIENCE, Issue 2 2002The somatostatin precursor. Abstract Synthetic peptides reproducing both the native domain around the dibasic cleavage site of pro-somatostatin, and mutated sequences thereof, previously assayed in site-directed mutagenesis experiments, have been studied by CD in different solvent systems, such as water, TFE/H2O, MeCN/H2O and aqueous SDS, in order to ascertain the ability of each solvent to stabilize secondary structural motifs. A combination of deconvolution methods and empirical calculations, that allow subtraction of the contributions due to unordered structures from the spectra, suggests that mainly two distinct families of ordered conformers containing ,-helix and/or structurally different ,-turns are present in solution, the relative stability of the different conformers depending on the nature of the solvent. The presence of ,-turns is in line with a previous NMR study in DMSO and DMSO/H2O. Comparison of the CD spectra in aqueous SDS of peptides undergoing processing with a sequence not processed in vivo shows that only the latter possesses a stable and detectable ,-helix population. This observation suggests that the structuration involving ,-turns but no ,-helix, which was observed by CD both in SDS and organic solvent/H2O mixtures at high water contents, might be of biological significance. The similarity of this structuration to molecular models obtained from NMR data in DMSO and DMSO/H2O is discussed. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] A New Method for the Treatment of Sperm Samples for Ultrastructural Study Based on the Use of Animal Tissues as Biological ContainersMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2007Concepción Junquera Abstract The study of the ultrastructure of spematozoa by means of transmission electron microscopy often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are: changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). To avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [source] EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Laurence Gall Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesisPROTEIN SCIENCE, Issue 9 2005Gábor Pál Abstract Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH. [source] Understanding dendritic cell biology and its role in immunological disorders through proteomic profilingPROTEOMICS - CLINICAL APPLICATIONS, Issue 2 2010Gabriela Bomfim Ferreira Abstract Dendritic cells (DC) have always been present on the bright spot of immune research. They have been extensively studied for the last 35 years, and much is known about their different phenotypes, stimulatory capacity, and role in the immune system. During the last 15 years, great attention has been given to studies on global gene and protein expression profiles during the differentiation and maturation processes of these cells. It is well understood that studying the proteome, together with information on the role of protein post-translational modifications (PTM), will reveal the real dynamics of a living cell. The rapid increase of proteomic studies during the last decade describing the differentiation and maturation process in DCs, as well as modifications brought by the use of different compounds that either increase or decrease their immunogenicity, reflects the importance of understanding the molecular processes behind the functional properties of these cells. In the present review, we will give an overview of proteomic studies focusing on DCs. Thereby we will concentrate on the importance of these studies in understanding DC behavior from a molecular point of view and how these findings have aided in understanding the differences in functional properties of these cells. [source] Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approachPROTEOMICS - CLINICAL APPLICATIONS, Issue 9 2008Gabriela Bomfim Ferreira Abstract Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14+ peripheral blood monocytes was investigated using two pH ranges (pH,4,7 and 6,9) (n,=,4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein-interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune-associated disorders. [source] Histone H1 and MAP Kinase Activities in Bovine Oocytes following Protein Synthesis InhibitionREPRODUCTION IN DOMESTIC ANIMALS, Issue 3-4 2001B Meinecke In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 ,g/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4,5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1,3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4,7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2,5 h earlier in comparison with untreated oocytes. [source] Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in ratsANDROLOGIA, Issue 2 2004A. Kapawa Summary. We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, andro-gen-binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke-exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation. [source] Morphological and morphometric attributes of epididymal and testicular spermatozoa following surgical sperm retrieval for obstructive and nonobstructive azoospermiaANDROLOGIA, Issue 6 2003Dr. S. Wood Summary. Whilst the morphological (shape) and morphometric (sperm head size) attributes of ejaculated spermatozoa have been well studied, the morphological and morphometric qualities of testicular and epididymal spermatozoa retrieved from males with obstructive and nonobstructive azoospermia is much less documented. We wished to examine the effect of aetiology of azoospermia and site of retrieval on the attributes of retrieved spermatozoa. This was a prospective observational study of 30 consecutive successful sperm retrievals, six for nonobstructive azoospermia and 24 for obstructive, of which five were retrieved from the epididymis and the remainder from the testis. The proportion of morphologically normal testicular spermatozoa in patients with obstructive and nonobstructive azoospermia was not significantly different (7% versus 7.6%, P = 0.97). Testicular spermatozoa from males with obstructive azoospermia showed an increase in frequency of sperm with small heads [47/180 (26%) versus 97/909 (11%), P = 0.036] as well as small acrosome and increasing vacuole formation over nonobstructive spermatozoa. Similarly, there was a significant increase in tail deformities and decreases in tail lengths in sperm from males with nonobstructive azoospermia. Epididymal spermatozoa showed significantly greater proportion of morphologically normal spermatozoa than testicular (20% versus 13%, P = 0.001) as well as a significant increase in acrosome vacuoles. Furthermore, morphometrically epididymal spermatozoa displayed with smaller head length, width and area than testicular spermatozoa. Testicular spermatozoa from obstructive azoospermia displayed significantly less tail defects (35% versus 57%, P = 0.003) as well as significantly longer tail lengths (30.6 ,m versus 10.7 ,m). These morphological and morphometric differences between epididymal and testicular and obstructive and nonobstructive spermatozoa may represent part of the natural maturation process. There were no associations between any morphological or morphometric abnormality with any significant parameter in subsequent use in ICSI. [source] Reduced sexual maturation in male post-smolt 1+ Atlantic salmon (Salmo salar L.) by dietary tetradecylthioacetic acidAQUACULTURE RESEARCH, Issue 5 2009Henriette Alne Abstract In the present study, the possible effect of dietary treatment on early sexual maturation in post-smolt Atlantic salmon, without any negative effect regarding growth, was investigated. The experiment was performed using 4400 individually marked (Pit tag) 1+ salmon, fed either a control diet or a diet supplemented with 0.5% tetradecylthioacetic acid (TTA) in duplicates for 3, 6 or 12 weeks after sea transfer. Compared with the control, dietary supplementation of TTA resulted in a threefold reduction in incidence of sexual mature males (0.6% vs. 1.8%). A curve-linear relationship between relative reduction in maturation and weeks of feeding TTA was found, indicating that the effect is most marked as a result of the first weeks of feeding and then levelling off. No negative dietary impact on growth was observed. As the level of fat in the muscle was reduced by dietary TTA, it seems that post-smolt supplemented dietary TTA do not accumulate high enough energy stores to start the maturation process, whereas the energy-enhancing effect of TTA due to increased fatty acid oxidation capacity may maintain the growth potential. Compared with immature salmon, sexually maturing fish revealed increased spring growth before the onset of maturation. [source] Theoretical Study of Catalytic Efficiency of a Diels,Alderase Catalytic Antibody: An Indirect Effect Produced During the Maturation ProcessCHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008Sergio Martí Dr. Abstract The Diels,Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39,A-11, that catalyze a Diels,Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate,protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate. [source] New developments in the production and use of stereoselective antibodiesCHIRALITY, Issue S1 2005Heike Hofstetter Abstract This article describes the production of stereoselective antibodies using both classical immunological and modern molecular biological techniques. Stereoselective antibodies against ,-hydroxy acids were raised in rabbits and mice and compared with previously produced anti-,-amino acid antibodies. It was found that both types of antibodies combine stereoselectivity with class-specificity. Sequence analyses revealed that antibodies with opposing stereoselectivities can be formed during the affinity maturation process from a common progenitor or independently using nonhomologous binding sites. For the first time, phage display was employed to obtain stereoselective antibody fragments. The versatility of stereoselective antibodies as chiral selectors was demonstrated by applying them in several immunosensors and in chiral chromatography. A simple, membrane-based optical sensor allowed detection of enantiomeric impurities at the 1/2,000 level (99.9% ee). Silica-based antibody chiral stationary phases could be used for enantiomer separation of aliphatic amino acids in standard-sized columns, while miniaturized columns allowed interfacing with an MS-detector. Chirality 17:S9,S18, 2005. © 2004 Wiley-Liss, Inc. [source] Gene expression studies in cultured dendritic cells: new indicators for the discrimination of skin sensitizers and irritants in vitroCLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2009S. Szameit Summary Background The replacement of animal tests for the detection of the sensitizing potential of chemicals is of great importance due to current legislation. One promising approach for the development of an in vitro assay is the exposure of immature dendritic cells (iDCs) to contact sensitizers and irritants, followed by an analysis of the maturation status of the cells. Objective The aim of this study was to further investigate the performance of our previously developed targeted microarray, the immune toxicity chip. In addition, we aimed to identify new marker genes for the discrimination of allergens and irritants using whole-genome microarrays. Methods Monocyte-derived iDCs were exposed to contact sensitizers and irritants in concentrations resulting in 10,20% cytotoxicity, as determined by dose,response curves. Changes in gene expression were analysed using the immune toxicity chip and a commercially available whole-genome microarray. Results Using the immune toxicity chip, we could identify a panel of marker genes suitable to discriminate strong allergens and irritants. Analysis with the whole-genome array revealed additional genes that are differentially expressed after allergen exposure, but not after irritant exposure. Hierarchical clustering of these genes showed distinct groups representing the different chemicals. Conclusion Here we show that our test system based on an immune-specific microarray is suitable for the discrimination of strong allergens and irritants. Genes detected as differentially expressed with the whole-genome array and previously not connected to the maturation process of DCs might be suitable candidate genes for the identification of weaker sensitizers. [source] Exploring Youth Development With Diverse Children: Correlates of Risk, Health, and Thriving BehaviorsJOURNAL FOR SPECIALISTS IN PEDIATRIC NURSING, Issue 1 2009Laureen H. Smith PURPOSE.,This study explored the relationships between internal and external assets, risk behaviors, health behaviors, and thriving behaviors in diverse children. DESIGN AND METHODS.,The strength of relationships existing between measures, differences between group means based on gender, grades earned, and school, and confidence interval (p , .05) were tested in a sample of 61 urban sixth graders. RESULTS.,Few assets were related to substance use. Assets were related to delinquency acts, health behaviors, and thriving indicators. Group differences between schools and gender and the total number of assets were noted. PRACTICE IMPLICATIONS.,Supporting assets are important to consider when nurses perform assessments and design interventions to support youths in their maturation processes. [source] Developmental changes in the ultrastructure of the lamprey lateral line nerve during metamorphosisJOURNAL OF MORPHOLOGY, Issue 7 2009S. Gelman Abstract The ultrastructure of the trunk lateral line nerve of larval and adult lampreys was studied with transmission electron microscopy. We confirmed that lampreys' lateral line nerve lacks myelin. Nevertheless, all axons were wrapped by Schwann cell processes. In the larval nerve, gaps between Schwann cells were observed, where the axolemma was covered only by a basal lamina, indicating an earlier developmental stage. In the adult nerve, glial (Schwann cell) ensheathment was mostly complete. Additionally, we observed variable ratios of axons to Schwann cells in larval and adult preparations. In the larval nerve, smaller axons were wrapped by one Schwann cell. Occasionally, a single Schwann cell surrounded two axons. Larger axons were associated with two to five Schwann cells. In the adult nerve, smaller axons were surrounded by one, but larger axons by three to eight Schwann cells. The larval epineurium contained large adipose cells, separated from each other by single fibroblast processes. This layer of adipose tissue was reduced in adult preparation. The larval perineurium was thin, and the fibroblasts, containing large amounts of glycogen granules, were arranged loosely. The adult perineurium was thicker, consisting of at least three layers of fibroblasts separated by collagen fibrils. The larval and adult endoneurium contained collagen fibrils oriented orthogonally to each other. Both larval and adult lateral line nerves possessed a number of putative fascicles weakly defined by a thin layer of perineurial fibroblasts. These results indicate that after a prolonged larval stage, the lamprey lateral line nerve is subjected to additional maturation processes during metamorphosis. J. Morphol. 2009. © 2009 Wiley-Liss, Inc. [source] Understanding dendritic cell biology and its role in immunological disorders through proteomic profilingPROTEOMICS - CLINICAL APPLICATIONS, Issue 2 2010Gabriela Bomfim Ferreira Abstract Dendritic cells (DC) have always been present on the bright spot of immune research. They have been extensively studied for the last 35 years, and much is known about their different phenotypes, stimulatory capacity, and role in the immune system. During the last 15 years, great attention has been given to studies on global gene and protein expression profiles during the differentiation and maturation processes of these cells. It is well understood that studying the proteome, together with information on the role of protein post-translational modifications (PTM), will reveal the real dynamics of a living cell. The rapid increase of proteomic studies during the last decade describing the differentiation and maturation process in DCs, as well as modifications brought by the use of different compounds that either increase or decrease their immunogenicity, reflects the importance of understanding the molecular processes behind the functional properties of these cells. In the present review, we will give an overview of proteomic studies focusing on DCs. Thereby we will concentrate on the importance of these studies in understanding DC behavior from a molecular point of view and how these findings have aided in understanding the differences in functional properties of these cells. [source] Mass spectrometry in the characterization of ambers.RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2008Amber is a fossil resin constituted of organic polymers derived through complex maturation processes of the original plant resin. A classification of eight samples of amber of different geological age (Miocene to Triassic) and geographical origin is here proposed using direct mass spectrometric techniques, i.e. laser desorption ionization (LDI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI), in order to obtain a fingerprint related to the amber origin. Differences and similarities were detected among the spectra with the four methods, showing quite complex spectra, full of ionic species in the mass range investigated (up to m/z 2000). The evaluation required statistical analysis involving multivariate techniques. Cluster analysis or principal component analysis (PCA) generally did not show a clear clustering with respect to the age of samples, except for the APPI method, which allowed a satisfying clustering. Using the total ion current (TIC) obtained by the different analytical approaches on equal quantities of the different amber samples and plotted against the age, the only significant correlation appeared to be that involving APPI. To validate the method, four amber samples from Cretaceous of Spain were analyzed. Also in this case a significant correlation with age was found only with APPI data. PCA obtained with TIC values from all the MS methods showed a fair grouping of samples, according to their age. Three main clusters could be detected, belonging to younger, intermediate and older fossil resins, respectively. This MS analysis on crude amber, either solid or extract, followed by appropriate multivariate statistical evaluation, can provide useful information on amber age. The best results are those obtained by APPI, indicating that the quantity of amber soluble components that can be photoionized decreases with increasing age, in agreement with the formation of highly stable, insoluble polymers. Copyright © 2008 John Wiley & Sons, Ltd. [source] Dendritic cells lentivirally engineered to overexpress interleukin-10 inhibit contact hypersensitivity responses, despite their partial activation induced by transduction-associated physical stressTHE JOURNAL OF GENE MEDICINE, Issue 3 2010Verena Besche Abstract Background Dendritic cells (DCs) constitute an attractive target for immunotherapeutic approaches. Because DCs are largely refractory to transfection with plasmid DNA, several viral transduction protocols were established. The potential side-effects of lentiviral transduction on the phenotype and activation state of DCs left unstimulated after transduction have not been assessed. There is a need to analyse these parameters as a result of the requirement of using DCs with a low activation state for therapeutic strategies intended to induce tolerance. Methods Lentivirally-transduced bone marrow (BM)-derived DCs (LV-DCs) in comparison with mock-transduced (Mock-DCs) and untreated DCs were analysed with regard to the induction of maturation processes on the RNA, protein and functional level. BM-DCs engineered to overexpress interleukin (IL)-10 were analysed for therapeutic potential in a mouse model of allergic contact dermatitis. Results Compared with untreated DCs, Mock-DCs and LV-DCs displayed an altered gene expression signature. Mock-DCs induced a stronger T cell proliferative response than untreated DCs. LV-DCs did not further augment the T cell proliferative response, but induced a slightly different T cell cytokine pattern compared to Mock-DCs. Accordingly, the gene promoter of the DC maturation marker fascin mediated efficient expression of the model transgene IL-10 in unstimulated-transduced BM-DCs. Nevertheless, IL-10 overexpressing BM-DCs exerted tolerogenic activity and efficiently inhibited the contact hypersensitivity response in previously hapten-sensitized mice. Conclusions Lentiviral transduction of BM-DCs results in their partial activation. Nevertheless, the transduction of these DCs with a vector encoding the immunomodulatory cytokine IL-10 rendered them tolerogenic. Thus, lentivirally-transduced DCs expressing immunomodulatory molecules represent a promising tool for induction of tolerance. Copyright © 2010 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||||||||||||||