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PubMed2006

Selection on protein-coding genes of natural cyanobacterial populations.

We examined the distribution of synonymous and non-synonymous changes in 12 protein-coding genes of natural populations of cyanobacteria to infer changes in gene functionality. By comparing mutation distributions within and across species using the McDonald-Kreitman test, we found data sets to contain evidence for purifying selection (hetR of Trichodesmium, nifH of Cylindrospermopsis raceborskii and rpoC1 of Anabaena lemmermannii) and positive selection (kaiC of Microcoleus chthonoplastes and rbcX of Anabaena and Aphanizomenon sp.). Other genes from the same set of clonal isolates (petB and rbcL in M. chthonoplastes and Anabaena/Aphanizomenon, respectively) did not harbour evidence for either form of selection. The results of branch models of codon evolution agreed fully with the results of the McDonald-Kreitman test in terms of significance and absolute value of the dN/dS estimates. The high frequency of gene-specific mutation patterns and their association with branches that separate closely related cyanobacterial genera suggest that evolutionary tests are suited to uncover gene-specific selective differentiation in cyanobacterial genomes. At the same time, given the lack of information about the history of cyanobacteria, analysis of larger numbers of protein-coding genes of clonal cyanobacterial isolates will produce more detailed pictures of the effects of natural selection.

Bacterial Proteins↗
PubMed2005

ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4 receptor-mediated signaling.

ICOS is the third member of the CD28 family molecules and plays a critical role in many T cell-dependent immune responses. Although accumulated data suggest that ICOS costimulatory signals play an important role in Th2-mediated immune responses, the molecular basis for this selective differentiation mechanism is largely unknown. To clarify this mechanism, we used DO11.10 TCR transgenic ICOS-/- mice and evaluated the nature of ICOS costimulatory signals during the process of Ag-specific activation and differentiation of naive CD4+ T cells. Results obtained from these experiments demonstrated that Ag stimulation of naive CD4+ T cells in the absence of an ICOS signal resulted in impaired Th2 development. Unlike previous reports, we found that primary IL-4 production by these T cells was intact and that IL-4R sensitivity of these T cells was reduced as evidenced by a profound defect in IL-4-induced Stat6 phosphorylation and the early induction of GATA-3. The fact that ICOS ligation of wild-type T cells significantly enhanced IL-4-induced Stat6 phosphorylation and primary GATA-3 induction, but not IL-4 transcription, of naive CD4+ T cells was consistent with the results obtained from ICOS-/- T cell experiments. These observations led us to propose that the predominant effect of ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4R-mediated signaling.

Adjuvants, Immunologic↗
PubMed1995

A genetical theory of species selection.

Species selection, differential rates of speciation or extinction resulting from species level characters, is often invoked as the main mechanism of macroevolution that is not simply an extension of microevolutionary processes. So long as we are careful in defining "species", the logic of species selection is sound. This does not mean, however, that this process can influence evolutionary dynamics under realistic conditions. The principal challenge to the efficacy of species selection as an evolutionary mechanism is the idea that selection between individuals within species will be so much more efficient as to swamp out any effects of selection between species. To assess this, a genetic model is constructed that includes simultaneous selection within and between species, and this is used to ask: under what conditions could species selection influence evolutionary dynamics, even in the face of opposing selection between individuals? The model shows that the efficacy of species selection is strongly determined by the time between speciation events (measured in individual generations), the mutation rate of the character under consideration, and the initial size of a newly formed reproductively isolated population. Data indicate that a few studied lineages have shown sufficiently high speciation rates to make species selection an important mechanism in the evolution of characters with mutation rates on the order of 10(-6) per generation. Quantitative characters, such as body size, generally change too readily for species selection to be relevant to their evolution. Complex characters, however, may be good candidates to be influenced by species selection. The interaction of selection within and between species can be subtle, with individual selection looking, from the standpoint of a species, very much like development of an individual. Furthermore, selection between individuals may be the main process assembling complex adaptations, while species selection allows them to persist over long periods of time.

Animals↗
PubMed1991

The cytokine receptor superfamily.

The binding of haemopoietic growth factors and cytokines to specific receptors triggers a cascade of intracellular events which results in cell proliferation and differentiation. The knowledge of ligand-receptor-signal pathways is not only important in understanding the pathophysiology of malignant disease but also essential for devising future therapeutic strategies. The advent of recombinant technology has made it possible to test the efficacy of selective differentiation therapy, and haemopoietic growth factors are undergoing clinical trials for a number of indications. In addition, increasingly the receptors for haemopoietic growth factors and cytokines have come under scientific scrutiny. Recently receptors for IL-2 alpha, IL-2 beta, IL-3, IL-4, IL-5, IL-6, IL-7, erythropoietin, G-CSF and GM-CSF have been isolated and cloned. It has become apparent that they have structural homology that is shared by receptors for growth hormone and prolactin, and this receptor group makes up the new cytokine receptor superfamily. The finding of sequence homology within these receptors suggests their evolutionary relationship. These receptors are transmembrane proteins 257-856 amino acids and their extracellular ligand-binding domain contains four conserved cysteine residues and a Trp-Ser-X-Trp-Ser motif. The secondary structure of the extracellular domain is made up of alpha-helices. High and low affinity binding forms exist for all these receptors. Binding affinity may depend on the formation of receptor heterodimers or multimers, association with other membrane proteins or differential glycosylation. Soluble receptor forms have been described for IL-2 alpha, IL-4, IL-5, IL-6 and IL-7. It is not known whether they are actively secreted or represent the degradation products of cell turnover. Their function may be to mop up excess cytokines and thereby confine the cytokine response. There is no sequence homology of the intracytoplasmic domains although several are rich in proline and serine residues, which may be important in mechanisms of signal transduction. No receptor in this superfamily functions as a receptor tyrosine kinase or has intrinsic protein tyrosine kinase activity. Detailed study of individual receptors holds clues to the regulation of receptor expression, ligand-receptor interactions and mechanisms involved in signal transduction. Such knowledge might explain the pleotropic effects cytokines may have on different cell types and their overlap in biological functions. Elevated levels of soluble IL-2 alpha receptor (Tac) are detected in hairy cell leukaemia, lymphomas and adult T-cell leukaemia (TL), and levels reflect tumour burden.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2002

Dynamics of bacterial phenotype selection in a colonized host.

The population dynamics of Helicobacter pylori during colonization in an infected animal host provide a quantifiable experimental model of in vivo microbial phenotype evolution. Phenotype variability in H. pylori populations can be typed as polymorphic expression of Lewis antigens on their cell surfaces. The high mutational frequency of H. pylori for Lewis expression provides substrate for differential selection by the host. Experimental challenge and successful colonization of mice and gerbils allows tracking of H. pylori phenotype variability from the initial inoculation to the ultimate establishment of a quasispecies. Colonization data provide a quantitative experimental model of phenotype evolution in a relatively large population (>10(4) individuals) over a relatively long evolutionary time scale (>10(3) generations). A mathematical model is developed to interpret the data in terms of the dynamic processes occurring during colonization. The mathematical model distinguishes the roles of selection and mutation; quantifies the effects of initial phenotype diversity, mutational frequency, and selective advantage; and applies generally to phenotype evolution in biological populations.

Animals↗
PubMed1996

Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/ elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs.

Blotting, Northern↗
PubMed2003

Estrogens reduce the expression of YKL-40 in the retina: implications for eye and joint diseases.

PURPOSE: To identify modifications in the gene expression profile of the ocular posterior segment in ovariectomized (OVX) mice with and without substitutive estradiol therapy and to select differentially expressed genes that could be relevant to the natural history of human age-related macular degeneration (AMD). METHODS: Chorioretinal tissues from two groups of 25 treated and untreated OVX mice were analyzed by using cDNA array technology. The expression level of selected genes was confirmed in triplicate by RT-PCR and related to the estrogenic status of the animals. Expression of the YKL-40 gene was further investigated in intact or diseased human retinas and in a murine model of experimental choroidal neovascularization (CNV), using laser pressure catapulting. RESULTS: Of the approximately 10,000 genes screened, only YKL-40 expression was significantly downregulated by 17-beta-estradiol. YKL-40 was expressed in intact human neural retina and in the RPE. The expression of YKL-40 was upregulated in experimental CNV and in neovascular membranes extracted from patients affected by the exudative form of AMD. CONCLUSIONS: These observations indicate that YKL-40 expression in the retina is modulated by serum levels of estradiol. This protein could be relevant to the development of AMD and is also a new mediator to take into account when evaluating the broad consequences of hormonal replacement therapy.

Adipokines↗
PubMed1999

A novel staining method for detecting phytase activity.

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

6-Phytase↗
PubMed1998

Antibody phage display technology and its applications.

In recent years, the use of display vectors and in vitro selection technologies has transformed the way in which we generate ligands, such as antibodies and peptides, for a given target. Using this technology, we are now able to design repertoires of ligands from scratch and use the power of phage selection to select those ligands having the desired (biological) properties. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. This review addresses recent progress in the construction of, and selection from phage antibody libraries, together with novel approaches for screening phage antibodies. As the quality of large naïve and synthetic antibody repertoires improves and libraries becomes more generally available, new and exciting applications are pioneered such as the identification of novel antigens using differential selection and the generation of receptor a(nta)gonists. A combination of the design and generation of millions to billions of different ligands, together with phage display for the isolation of binding ligands and with functional assays for identifying (and possibly selecting) bio-active ligands, will open even more challenging applications of this inspiring technology, and provide a powerful tool for drug and target discovery well into the next decade.

Animals↗
PubMed1989

A novel MHC class II epitope expressed in thymic medulla but not cortex.

The repertoire of receptors expressed by peripheral T cells is the result of two selective events that occur during intrathymic development. Positive selection expands cells able to recognize foreign peptides presented by self MHC molecules, and negative selection eliminates cells reactive to self MHC molecules and associated self peptides. Chimaera studies suggest that, at least in the case of T cells recognizing MHC class II, interaction with thymic cortical epithelial cells is responsible for the former, whereas thymic medullary cells, of bone marrow origin, mediate the latter. This view of thymic development is supported by recent morphometric analyses, showing that autoreactive cells are found in thymic cortex but not medulla. Although numerous studies have shown that MHC class II molecules are expressed in both sites, none provides any explanation for the differential selection of T cells that is observed. Here, we describe a novel MHC class II epitope which is found on cells in thymic medulla but not cortex. The antibody to this epitope reacts with about 10% of class II molecules on B cells and may be recognizing a self peptide-MHC complex. These results provide the first evidence for differential expression of class II epitopes in different tissues and are compatible with the hypothesis that different ligands, rather than different affinity thresholds for the same ligand, are involved in positive and negative selection of the T-cell repertoire.

Animals↗
PubMed1991

Pharmacological characteristics of NP-252, a new dihydropyridine slow Ca2+ channel blocker, in isolated rabbit vascular smooth muscle and guinea pig myocardium: vascular selectivity.

NP-252, a new dihydropyridine derivative, and nifedipine non-competitively inhibited contractile responses to KCl and responses to Ca2+ in Ca(2+)-free medium containing KCl in rabbit aorta and renal, mesenteric, coronary and basilar arteries, mesenteric veins and vena cava. The effects of NP-252 in these smaller arteries and veins were much greater than those in aorta. However, a similar differential selectivity was not seen with nifedipine. In aorta, only NP-252 reduced total La3+ resistant 45Ca binding. However, the increases in bound 45Ca at La3+ resistant sites and 45Ca unidirectional influx due to KCl were inhibited by both NP-252 and nifedipine. The displacement of [3H]nitrendipine binding to rabbit aorta was monophasic for both NP-252 and nifedipine. In guinea-pig papillary muscles, NP-252 (greater than 10(-7) M) slightly decreased action potential duration, developed tension and slow action potentials. The cardiac effects of NP-252 were much less prominent than those of nifedipine. These results indicate that NP-252 inhibits voltage-operated Ca2+ channels in small arteries and veins much more effectively than those in aorta, and this tissue selectivity is more apparent for NP-252 than nifedipine.

Action Potentials↗
PubMed1982

Selective inhibitors of platelet arachidonic acid metabolism: aggregation independent of lipoxygenase.

The use of acetylenic acid analogs differing in chain length or position of the triple bonds permitted the systemic study of structure activity relationships for the arachidonate metabolizing enzymes (i.e, cyclo-oxygenase and lipoxygenase) in platelets and the relationship of these enzymes to aggregation. We were able to demonstrate analogs that were differentially selective in altering platelet arachidonic acid metabolism. We found analogs that preferentially: (a) inhibited cyclo-oxygenase only, (b) inhibited the 12-lipoxygenase only, (c) inhibited the cyclo-oxygenase and lipoxygenase, and (d) inhibited neither enzyme in platelets. There was a direct correlation between the rank order of potency of the acetylenic analogs to inhibit platelet cyclo-oxygenase and to suppress aggregation. Certain structural features of the triynoic acetylenic analogs were critical in influencing platelet function; thus the presence of a triple bond at position 14 as well as the lack of triple bond at position 5 resulted in analogs which inhibited both cyclo-oxygenase and platelet aggregation. On the other hand, analogs that inhibited only platelet 12-lipoxygenase were very weak inhibitors of platelet aggregation. These inhibitors provide potentially powerful tools for dissociating the two arachidonate metabolic pathways. If other tissues are are readily manipulated as platelets, the analogs may be especially useful for gaining insight into the contribution of lipoxygenase products to biological function.

Arachidonate Lipoxygenases↗
PubMed1995

Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.

Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon gamma (IFN-gamma) activation sites, which were first identified in the promoters of IFN-gamma-inducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-gamma, interleukin (IL) 6, granulocyte-macrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT--AA motif with a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.

Base Sequence↗
PubMed2002

Development of CD25(+) T cells secreting transforming growth factor-beta1 by altered peptide ligands expressed as self-antigens.

This study demonstrates that CD4(+) T cells specific for an altered self-antigen differentiate to T cells secreting transforming growth factor (TGF)-beta1. In this study, we utilized mice expressing an altered peptide ligand containing a single amino acid substitution of moth cytochrome c 88-103 peptide. In these mice, antigen-specific T cells escaping thymic negative selection differentiated into T cells with an effector/memory phenotype, CD44(high), CD45RB(low), CD62L(-) and CD25(intermediate). The expression of CD25 and high levels of CD44 was initiated in the thymus during the development from CD4(+)CD8(+) to CD4(+); a large proportion of maturing CD4(+) thymocytes expressed both CD25 and high levels of CD44. Upon antigen stimulation, CD4(+) T cells derived from these mice did not proliferate or secrete IL-2, but secreted TGF-beta1. Neutralizing antibodies to TGF-beta1 reversed the impaired proliferative responses to the antigen, suggesting that TGF-beta1 secreted from these T cells negatively regulates T cell responses.

Amino Acid Sequence↗
PubMed2006

Differential expression of profibrotic and growth factors in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is a multifactorial process, where both immunological and nonimmunological factors play roles. Microarrays detect thousands of genes simultaneously. METHODS: We have analyzed gene expression profiles of 16 kidney transplant biopsy samples with CAN by high-density oligonucleotide microarrays, comparing to six normal transplant biopsies. Eight CAN biopsies showed nodular arteriolar hyalinization and one was positive for C4d staining. RESULTS: Hierarchical clustering analysis of the 22 biopsies revealed differential gene expression patterns in CAN versus the control biopsies. However, microarray analysis did not reveal differential gene expression patterns in patients with or without arteriolar hyalinization. Fifty percent of the 100 genes with highest hybridization intensities in a C4d positive sample were related to cellular and humoral immune response. Although 212 genes were upregulated a minimum of 1.5-fold, 112 genes were downregulated in CAN samples. There was differential expression of profibrotic and growth factors that while transforming growth factor-beta induced factor, thrombospondin 1, and platelet derived growth factor-C were up-regulated, vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factors 1 and 9 were downregulated. Selected differentially expressed genes were confirmed in microdissected samples by real-time quantitative PCR. Immunopathologic examination of biopsies revealed strong TGF-beta but decreased glomerular VEGF expression in CAN. CONCLUSION: Microarrays might be an important tool to uncover the mechanisms of multifactorial diseases, such as CAN.

Adult↗
PubMed2006

Reproductive success of bromadiolone-resistant rats in absence of anticoagulant pressure.

Resistance to anticoagulant rodenticides in brown rats (Rattus norvegicus Berk.) is associated with pleiotropic effects, notably with an increased dietary vitamin K requirement. Owing to this disadvantage, resistance is believed to be selected against if anticoagulant selection is absent. In small experimental populations of wild brown rats, an investigation was carried out to establish whether tolerance to anticoagulant exposure changed over a period of 2 years. In the same populations, DNA microsatellite markers were used to infer parentage, and this made it possible to estimate reproductive success of sensitive and resistant rats and estimate effective population size, Ne. Even though there was evidence for a selection against resistant rats with high vitamin K requirement, anticoagulant tolerance was not seen to be significantly influenced in the absence of bromadiolone selection. As the population size under investigation was small, random genetic drift may have played a role in this. In the presence of bromadiolone selection, however, the tolerance was significantly increased, suggesting that continuous selection will increase the proportion of highly resistant rats in the population. It was found that, for both males and females, surprisingly few individuals contributed to the next generation with numerous offspring, and most breeders contributed with none or a single offspring. The expected higher reproductive success and consequent increase in proportional numbers of sensitive rats in the absence of anticoagulant selection could not be observed. Among the resistant rats, moderately resistant females were found to be better breeders than highly resistant breeders, but for resistant males the reverse was true. This could be explained by the fact that the increased vitamin K requirement results in sex differential selection; in highly resistant males the selection presumably takes place at the immature stage, whereas in females the vitamin K requirement becomes crucial at the reproductive stage, as vitamin K is not only essential for the blood clotting process but also for bone formation.

4-Hydroxycoumarins↗
PubMed1995

The reduction principle for recombination under density-dependent selection.

In diploid random mating populations with constant viability selection, genetic modifiers of recombination, introduced near equilibria that exhibit genetic association, invade if they reduce recombination. In this study we combine ecological and standard populations genetics in a haploid multilocus model that includes density-dependent regulation of population size and weak density-dependent differential selection among the multilocus genotypes. An allele that affects recombination among the genes contributing to the ecological selection, introduced near a stable equilibrium of the ecological-genetic system, invades if it reduces a weighted average of the recombination rates among pairs of loci under selection. This generalizes the Reduction Principle for the evolution of recombination (M. W. Feldman and U. Liberman, Proc. Nat. Acad. Sci. USA 83, 4824-4827, 1986; L. A. Zhivotovsky, W. M. Feldman, and F. B. Christiansen, Theor. Popul. Biol. 44, 225-245, 1993). It is also shown that the stronger the extent of density-dependence, the weaker the selection for reduced recombination.

Alleles↗
PubMed2006

Genes differentially expressed in oropharyngeal tonsils and mandibular lymph nodes of tuberculous and nontuberculous European wild boars naturally exposed to Mycobacterium bovis.

Bovine tuberculosis, caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle worldwide. The bacterium infects other animal species, both domesticated and wild, and this range of hosts complicates attempts to control or eradicate the disease. Despite advances in the characterization of the mechanisms involved in host-pathogen interactions and host cell responses to M. tuberculosis complex in human, bovine and mouse cells, differentially expressed genes in tissue biopsies of naturally occurring tuberculous and nontuberculous exposed individuals have been poorly characterized. In this study, differential gene expression was analysed using suppression-subtractive hybridization in oropharyngeal tonsils and mandibular lymph nodes of field-collected tuberculous and nontuberculous European wild boars from a tuberculosis-endemic area of Spain. Real-time PCR and semiquantitative reverse-transcriptase PCR of selected genes confirmed the results of the suppression-subtractive hybridization analysis. Protein expression of selected differentially expressed genes was analysed by radial immunodiffusion or immunohistochemistry. Differential gene expression varied among tuberculous and non-tuberculous groups and between tonsils and lymph nodes. Single and multiple cellular mechanisms were affected, including signal transduction, immune response, inflammation, stress, apoptosis/antiapoptosis, cell structure, adhesion and transport, protein and DNA/RNA metabolism and enzymatic processes. These results demonstrate the modulation of gene expression by mycobacterial infection in tonsils and mandibular lymph nodes of European wild boars naturally exposed to M. bovis, and provide a basis for defining host-pathogen interactions and the mechanism of protective immunity.

Animals↗