rapid-fractionation-isolation-whole-blood-components-samples-obtained

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

University of Illinois at Urbana-Champaign

The mechanism responsible for glucocorticoid receptor (GR)-mediated induction of rat hepatic hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription was investigated. We previously reported that the region of the SULT2A-40/41 5'-flanking region delimited by -158 to -77 nucleotides relative to the transcription start site was sufficient to support GR-inducible expression. This region of the SULT2A-40/41 gene does not contain a consensus glucocorticoid receptor-responsive element, but does contain two consensus sites for liver-enriched CCAAT/enhancer-binding protein (C/EBP) transcription factors. In the present study, incubation of primary cultured rat hepatocytes with a GR-activating concentration (10(-7) M) of a potent glucocorticoid, dexamethasone or triamcinolone acetonide (TA), rapidly produced increases in C/EBPalpha and C/EBPbeta nuclear protein contents, as measured by Western blot or in vitro DNA-binding activity analysis, that preceded increases in SULT2A-40/41 mRNA and protein levels. Transient cotransfection of SULT2A-40/41 reporter plasmids with a dominant negative C/EBP expression plasmid completely blocked TA-inducible SULT2A-40/41 reporter gene expression. Linker scanning and site-directed mutagenesis of the proximal SULT2A-40/41 5'-flanking region, complemented by in vitro DNA-binding analyses, indicated that the more distal C/EBP site was important for controlling SULT2A-40/41 promoter activity. These data support a role for GR-inducible C/EBPalpha and C/EBPbeta expression in the transactivation of hepatic SULT2A-40/41 expression.

Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: role of CCAAT/enhancer-binding protein liver-enriched transcription factors.

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver.

The cytochrome P450 expression profile was determined in the MCF10A human breast epithelial cell line, as was the ability of this cell line to catalyze the bioactivation of the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Using non-quantitative reverse transcription-polymerase chain reaction (RT-PCR), transcripts for CYP1B1, CYP2J2, CYP2R1, CYP2U1, CYP2W1, CYP4B1, CYP4F, CYP4V2, CYP4X1 and CYP4Z1 were detected in both sub-confluent and confluent MCF10A cells. By contrast, CYP1A2 mRNA was detected only in confluent MCF10A cells, while CYP1A1, CYP2S1 and CYP2F1 were detected predominantly or exclusively in sub-confluent cultures. 2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment of confluent MCF10A cells markedly induced microsomal ethoxyresorufin O-deethylase activity and CYP1A1, CYP1A2 and CYP1B1 mRNA levels, as determined by real-time RT-PCR, while treatment with 10(-4)M PhIP had little effect on these P450 transcript levels. Treatment of confluent MCF10A cells with PhIP (10(-4)M) for 24, 48 or 72 h produced time-dependent increases in the amounts of DNA adducts, as measured by (32)P-post-labeling. These results indicate that multiple P450s, including those known to catalyze PhIP N-oxidation, are expressed in MCF10A cells, and that this non-neoplastic human breast epithelial cell line contains sufficient enzymatic machinery to support PhIP bioactivation and generate DNA damage.

Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells.

Galectin-1 is an anti-inflammatory lectin with pleiotropic regulatory functions at the crossroads of innate and adaptive immunity. It is expressed in immune privileged sites and is implicated in establishing maternal-fetal immune tolerance, which is essential for successful pregnancy in eutherian mammals. Here, we show conserved placental localization of galectin-1 in primates and its predominant expression in maternal decidua. Phylogenetic footprinting and shadowing unveil conserved cis motifs, including an estrogen responsive element in the 5' promoter of LGALS1, that were gained during the emergence of placental mammals and could account for sex steroid regulation of LGALS1 expression, thus providing additional evidence for the role of galectin-1 in immune-endocrine cross-talk. Maximum parsimony and maximum likelihood analyses of 27 publicly available vertebrate and seven newly sequenced primate LGALS1 coding sequences reveal that intense purifying selection has been acting on residues in the carbohydrate recognition domain and dimerization interface that are involved in immune functions. Parsimony- and codon model-based phylogenetic analysis of coding sequences show that amino acid replacements occurred in early mammalian evolution on key residues, including gain of cysteines, which regulate immune functions by redox status-mediated conformational changes that disable sugar binding and dimerization, and that the acquired immunoregulatory functions of galectin-1 then became highly conserved in eutherian lineages, suggesting the emergence of hormonal and redox regulation of galectin-1 in placental mammals may be implicated in maternal-fetal immune tolerance.

Emergence of hormonal and redox regulation of galectin-1 in placental mammals: implication in maternal-fetal immune tolerance.

Galectins are proteins that regulate immune responses through the recognition of cell-surface glycans. We present evidence that 16 human galectin genes are expressed at the maternal-fetal interface and demonstrate that a cluster of 5 galectin genes on human chromosome 19 emerged during primate evolution as a result of duplication and rearrangement of genes and pseudogenes via a birth and death process primarily mediated by transposable long interspersed nuclear elements (LINEs). Genes in the cluster are found only in anthropoids, a group of primate species that differ from their strepsirrhine counterparts by having relatively large brains and long gestations. Three of the human cluster genes (LGALS13, -14, and -16) were found to be placenta-specific. Homology modeling revealed conserved three-dimensional structures of galectins in the human cluster; however, analyses of 24 newly derived and 69 publicly available sequences in 10 anthropoid species indicate functional diversification by evidence of positive selection and amino acid replacements in carbohydrate-recognition domains. Moreover, we demonstrate altered sugar-binding capacities of 6 recombinant galectins in the cluster. We show that human placenta-specific galectins are predominantly expressed by the syncytiotrophoblast, a primary site of metabolic exchange where, early during pregnancy, the fetus comes in contact with immune cells circulating in maternal blood. Because ex vivo functional assays demonstrate that placenta-specific galectins induce the apoptosis of T lymphocytes, we propose that these galectins reduce the danger of maternal immune attacks on the fetal semiallograft, presumably conferring additional immune tolerance mechanisms and in turn sustaining hemochorial placentation during the long gestation of anthropoid primates.

A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death.

In anthropoid primates, growth hormone (GH) genes have undergone at least 2 independent locus expansions, one in platyrrhines (New World monkeys) and another in catarrhines (Old World monkeys and apes). In catarrhines, the GH cluster has a pituitary-expressed gene called GH1; the remaining GH genes include placental GHs and placental lactogens. Here, we provide cDNA sequence evidence that the platyrrhine GH cluster also includes at least 3 placenta expressed genes and phylogenetic evidence that placenta expressed anthropoid GH genes have undergone strong adaptive evolution, whereas pituitary-expressed GH genes have faced strict functional constraint. Our phylogenetic evidence also points to lineage-specific gene gain and loss in early placental mammalian evolution, with at least three copies of the GH gene present at the time of the last common ancestor (LCA) of primates, rodents, and laurasiatherians. Anthropoid primates and laurasiatherians share gene descendants of one of these three copies, whereas rodents and strepsirrhine primates each maintain a separate copy. Eight of the amino-acid replacements that occurred on the lineage leading to the LCA of extant anthropoids have been implicated in GH signaling at the maternal-fetal interface. Thus, placental expression of GH may have preceded the separate series of GH gene duplications that occurred in catarrhines and platyrrhines (i.e., the roles played by placenta-expressed GHs in human pregnancy may have a longer evolutionary history than previously appreciated).

Ancient origin of placental expression in the growth hormone genes of anthropoid primates.

The phylogenetic position of tarsiers within the primates has been a controversial subject for over a century. Despite numerous morphological and molecular studies, there has been weak support for grouping tarsiers with either strepsirrhine primates in a prosimian clade or with anthropoids in a haplorrhine clade. Here, we take advantage of the recently released whole genome assembly of the Philippine tarsier, Tarsius syrichta, in order to infer the phylogenetic relationship of Tarsius within the order Primates. We also present estimates of divergence times within the primates. Using a 1.26 million base pair multiple sequence alignment derived from 1078 orthologous genes, we provide overwhelming statistical support for the presence of a haplorrhine clade. We also present divergence date estimates using local relaxed molecular clock methods. The estimated time of the most recent common ancestor of extant Primates ranged from 64.9 Ma to 72.6 Ma, and haplorrhines were estimated to have a most recent common ancestor between 58.9 Ma and 68.6 Ma. Examination of rates of nucleotide substitution in the three major extant primate clades show that anthropoids have a slower substitution rate than either strepsirrhines or tarsiers. Our results provide the framework on which primate morphological, reproductive, and genomic features can be reconstructed in the broader context of mammalian phylogeny.

Genomic data reject the hypothesis of a prosimian primate clade.

The chorioallantoic placenta connects mother and fetus in eutherian pregnancies. In order to understand the evolution of the placenta and provide further understanding of placenta biology, we sequenced the transcriptome of a term placenta of an African elephant (Loxodonta africana) and compared these data with RNA sequence and microarray data from other eutherian placentas including human, mouse, and cow. We characterized the composition of 55,910 expressed sequence tag (i.e., cDNA) contigs using our custom annotation pipeline. A Markov algorithm was used to cluster orthologs of human, mouse, cow, and elephant placenta transcripts. We found 2,963 genes are commonly expressed in the placentas of these eutherian mammals. Gene ontology categories previously suggested to be important for placenta function (e.g., estrogen receptor signaling pathway, cell motion and migration, and adherens junctions) were significantly enriched in these eutherian placenta-expressed genes. Genes duplicated in different lineages and also specifically expressed in the placenta contribute to the great diversity observed in mammalian placenta anatomy. We identified 1,365 human lineage-specific, 1,235 mouse lineage-specific, 436 cow lineage-specific, and 904 elephant-specific placenta-expressed (PE) genes. The most enriched clusters of human-specific PE genes are signal/glycoprotein and immunoglobulin, and humans possess a deeply invasive human hemochorial placenta that comes into direct contact with maternal immune cells. Inference of phylogenetically conserved and derived transcripts demonstrates the power of comparative transcriptomics to trace placenta evolution and variation across mammals and identified candidate genes that may be important in the normal function of the human placenta, and their dysfunction may be related to human pregnancy complications.

Elephant transcriptome provides insights into the evolution of eutherian placentation.

In comparison with other primate species, humans have an extended juvenile period during which the brain is more plastic. In the current study we sought to examine gene expression in the cerebral cortex during development in the context of this adaptive plasticity. We introduce an approach designed to discriminate genes with variable as opposed to uniform patterns of gene expression and found that greater inter-individual variance is observed among children than among adults. For the 337 transcripts that show this pattern, we found a significant overrepresentation of genes annotated to the immune system process (pFDR ~/= 0). Moreover, genes known to be important in neuronal function, such as brain-derived neurotrophic factor (BDNF), are included among the genes more variably expressed in childhood. We propose that the developmental period of heightened childhood neuronal plasticity is characterized by more dynamic patterns of gene expression in the cerebral cortex compared to adulthood when the brain is less plastic. That an overabundance of these genes are annotated to the immune system suggests that the functions of these genes can be thought of not only in the context of antigen processing and presentation, but also in the context of nervous system development.

Dynamic gene expression in the human cerebral cortex distinguishes children from adults.

Human brain development follows a unique pattern characterized by a prolonged period of postnatal growth and reorganization, and a postnatal peak in glucose utilization. The molecular processes underlying these developmental changes are poorly characterized. The objectives of this study were to determine developmental trajectories of gene expression and to examine the evolutionary history of genes differentially expressed as a function of age.

Characterization of human cortical gene expression in relation to glucose utilization.

The human brain and human cognitive abilities are strikingly different from those of other great apes despite relatively modest genome sequence divergence. However, little is presently known about the interspecies divergence in gene structure and transcription that might contribute to these phenotypic differences. To date, most comparative studies of gene structure in the brain have examined humans, chimpanzees, and macaque monkeys. To add to this body of knowledge, we analyze here the brain transcriptome of the western lowland gorilla (Gorilla gorilla gorilla), an African great ape species that is phylogenetically closely related to humans, but with a brain that is approximately one-third the size. Manual transcriptome curation from a sample of the planum temporale region of the neocortex revealed 12 protein-coding genes and one noncoding-RNA gene with exons in the gorilla unmatched by public transcriptome data from the orthologous human loci. These interspecies gene structure differences accounted for a total of 134 amino acids in proteins found in the gorilla that were absent from protein products of the orthologous human genes. Proteins varying in structure between human and gorilla were involved in immunity and energy metabolism, suggesting their relevance to phenotypic differences. This gorilla neocortical transcriptome comprises an empirical, not homology- or prediction-driven, resource for orthologous gene comparisons between human and gorilla. These findings provide a unique repository of the sequences and structures of thousands of genes transcribed in the gorilla brain, pointing to candidate genes that may contribute to the traits distinguishing humans from other closely related great apes. J. Comp. Neurol., 2015. © 2015 Wiley Periodicals, Inc.

Article	Year
Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: role of CCAAT/enhancer-binding protein liver-enr... Drug metabolism and disposition: the biological fate of chemicals\| PubMed ID: 15502011	2005
Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 16339912	2006
Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells. Chemico-biological interactions\| PubMed ID: 16527260	2006
Emergence of hormonal and redox regulation of galectin-1 in placental mammals: implication in maternal-fetal immune tolerance. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18824694	2008
A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19497882	2009
Ancient origin of placental expression in the growth hormone genes of anthropoid primates. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19805162	2009
Genomic data reject the hypothesis of a prosimian primate clade. Journal of human evolution\| PubMed ID: 21620437	2011
Elephant transcriptome provides insights into the evolution of eutherian placentation. Genome biology and evolution\| PubMed ID: 22546564	2012
Dynamic gene expression in the human cerebral cortex distinguishes children from adults. PloS one\| PubMed ID: 22666384	2012
Characterization of human cortical gene expression in relation to glucose utilization. American journal of human biology : the official journal of the Human Biology Council\| PubMed ID: 23559490	2013
High-throughput RNA sequencing reveals structural differences of orthologous brain-expressed genes between western lowland gorillas and humans. The Journal of comparative neurology\| PubMed ID: 26132897	2015

Amy Weckle

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