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At least 919 records · Page 51Linked to original sources
PubMed2006

The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells.

The Tec family tyrosine kinases, Itk and Rlk, are expressed in thymocytes and peripheral T cells and regulate thresholds of T cell receptor signaling. Yet little is known about the specific role of Itk- and Rlk-dependent signals in CD8(+) T cell maturation. We show here that Itk(-/-) and Rlk(-/-)Itk(-/-) mice were nearly devoid of conventional CD8(+) T cells and, instead, contained a large population of CD8(+) T cells that bear striking similarity to lineages of innate lymphocytes. Itk(-/-) and Rlk(-/-)Itk(-/-) CD8(+) thymocytes and T cells were CD44(hi), CD122(+), and NK1.1(+); were able to produce interferon-gamma directly ex vivo; and were dependent on interleukin-15. Itk(-/-) and Rlk(-/-)Itk(-/-) CD8(+) thymocytes expressed abundant transcripts for the T box transcription factor, eomesodermin, correlating with their phenotype and function. These data indicate a critical role for Itk and Rlk in conventional CD8(+) T cell development in the thymus.

Animals↗
PubMed2026

Tonsillar expression quantitative trait loci verify and expand genetic contributors to childhood atopic diseases.

BACKGROUND: The spectrum of causal variants, mechanisms, and immunologic gene networks that influence pediatric atopic traits is not completely understood. Human genetic variation associated with transcript abundance (expression quantitative trait loci [eQTLs]) can help to advance our understanding, yet prior work has focused on profiling immune cell populations collected from peripheral blood primarily in adult populations, leaving tissue-resident lymphocytes collected from children uncharacterized. OBJECTIVE: We sought to characterize gene expression of 4 populations of tonsil-derived immune cell types collected from pediatric patients. METHODS: We collected naive B, germinal center B, naive T, and T follicular helper cells from the discarded tonsils of 103 children across development (age range 1-19). Following genotyping and RNA sequencing of samples, we performed differential expression and eQTL analysis, then statistically linked eQTL signals to relevant atopic traits via colocalization. RESULTS: We found differentially expressed genes across cell types and identified 13,393 expression genes (eGenes) (1,793 eGenes not previously reported in similar datasets) influenced by 27,603 eQTLs (5,199 eQTLs not previously reported). We linked eQTLs to associations identified in pediatric and adult asthma and atopy traits, nominating 78 eGenes including TRAF3, ZBTB10, and JAZF1 in disease-relevant cell types. CONCLUSIONS: Our freely available resource exemplifies the importance of discovery in native tissues and across human development.

Expression quantitative trait locus↗
PubMed2005

Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue.

An optimized analytical expression profiling strategy based on gel-free multidimensional protein identification technology (MudPIT) is reported for the systematic investigation of biochemical (mal)-adaptations associated with healthy and diseased heart tissue. Enhanced shotgun proteomic detection coverage and improved biological inference is achieved by pre-fractionation of excised mouse cardiac muscle into subcellular components, with each organellar fraction investigated exhaustively using multiple repeat MudPIT analyses. Functional-enrichment, high-confidence identification, and relative quantification of hundreds of organelle- and tissue-specific proteins are achieved readily, including detection of low abundance transcriptional regulators, signaling factors, and proteins linked to cardiac disease. Important technical issues relating to data validation, including minimization of artifacts stemming from biased under-sampling and spurious false discovery, together with suggestions for further fine-tuning of sample preparation, are discussed. A framework for follow-up bioinformatic examination, pattern recognition, and data mining is also presented in the context of a stringent application of MudPIT for probing fundamental aspects of heart muscle physiology as well as the discovery of perturbations associated with heart failure.

Animals↗
PubMed2004

Vitamin D receptor B1 and exon 1d: functional and evolutionary analysis.

The vitamin D receptor (VDR) shares a conserved structural and functional organization with other nuclear receptor (NR) superfamily members. For many NRs, N-terminal variant isoforms that display distinct cell-, stage- and promoter-specific actions have been identified. The novel VDR isoform VDRB1, with a 50 amino acid N-terminal extension, is produced from low abundance transcripts that contain exon 1d of the human VDR locus. There is evidence for the conservation of this exon in other mammalian and avian species. The transactivation differences between VDRB1 and the original VDR, clarified here, provide insights into mechanisms that may contribute to functional differences and potentially distinct physiological roles for these two VDR isoforms.

Animals↗
PubMed2006

In vivo and in vitro CYP1B mRNA expression in channel catfish.

Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to AhR ligands. The full-length catfish CYP1B cDNA is 2417 nt to the polyA tail and encodes a putative protein of 536 amino acids. It has 67% amino acid similarity to carp and zebrafish CYP1B and 68% similarity to carp CYP1B2. Male channel catfish were collected from three Mississippi Delta sites: Lake Roebuck, Itta Bena; Bee Lake, Thornton; and Sunflower River, Indianola. Total RNA was isolated from wild-caught catfish gill, blood, gonad and liver tissues. Quantitative real-time reverse transcriptase PCR was used to determine relative induction of CYP1B in wild catfish compared to laboratory control and BaP-exposed catfish (20mg/kg i.p. after 4 days). BaP exposure significantly induced CYP1B message in blood, gonad, and liver of laboratory catfish. In these same tissues of wild catfish from sites with relatively low sediment contaminants, CYP1B message was not statistically increased relative to laboratory control catfish. CYP1B transcript abundance was higher in gills compared to other tissues in both laboratory and wild catfish. When primary cultured gill cells were treated with increasing concentrations of BaP, TCDD, and PCBs 77, 126 and 169, CYP1B mRNA was induced more than 10-fold while PCB153 and 4,4'DDT did not cause significant CYP1B induction. Our results suggest that catfish CYP1B is induced by the classic AhR ligands.

Animals↗
PubMed2004

A collection of bovine cDNA probes for gene expression profiling in muscle.

Array technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to muscle development and pathology as well as meat production in livestock species have been hampered by the lack of appropriate cDNA collections. To overcome this problem, a directed cDNA library was constructed starting from 23 muscles of meat-producing bovines to derive a collection of 3573 clones. A preliminary sequence characterization of this collection indicated that the most abundant transcripts correspond to genes encoding proteins involved in energy metabolism (COX and NADH dehydrogenase subunits) and belonging to the contractile apparatus (myosin chains and troponin isoforms). From this cDNA library, we selected a set of 435 clones representing 340 unique genes, of which 24 were novel. This collection was subsequently completed with 75 specific cDNA probes for genes of interest already studied in our laboratory. The bovine 'muscle' cDNA repertoire thus designed was spotted onto a nylon membrane (macroarray) in order to test its utility to further investigate the transcriptome of bovine muscles in relation to meat quality traits. It is also anticipated that this type of collection might be useful for the study of chronic myologic diseases in other mammalian species, including humans.

Animals↗
PubMed2004

Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.

As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.

Animals↗
PubMed2006

Changes in immune gene expression and resistance to bacterial infection in lobster (Homarus gammarus) post-larval stage VI following acute or chronic exposure to immune stimulating compounds.

Real-time PCR was used to measure changes in transcript abundance of genes encoding important immune proteins, namely prophenoloxidase (proPO gene), beta-1,3-glucan binding protein (betaGBP gene) and a 12.2 kDa antimicrobial peptide (amp gene) in post-larval stage VI (PLVI) juveniles of the European lobster, Homarus gammarus. Gene expression was studied in both healthy PLVI and following single or repeat exposure to a range of compounds claimed to induce immune reactivity. A single acute (3-h) exposure to any of the tested stimulants did not produce a significant increase in expression of either the proPO or betaGBP genes, measured 6h after stimulation. However, there were a small sub-group of positive responders, identified mainly from betaGBP expression, within the experimental groups stimulated with either a beta-1,3-glucan or an alginate. There was also no significant increase in the expression of any of the three genes tested 24 h after repeated weekly (3-h) exposures to a either the beta-1,3-glucan or the alginate over the longer (36-day) period. The results do show that amp is expressed at an extremely high level compared to proPO or betaGBP in healthy animals and a significant correlation was found between the expression of proPO and both betaGBP and amp, irrespective of whether or not the larvae were stimulated. None of the immune stimulated compounds improved survival of PLVI challenged with the opportunistic pathogen, Listonella anguillarum, or the lobster pathogen, Aerococcus viridans var. homari. Thus, we found no evidence to support recent claims that immunity and disease resistance can be primed or promoted within a given population of crustaceans or that these animals exhibit functional immune memory to some soluble immune elicitors.

Animals↗
PubMed2005

Robust dysregulation of gene expression in substantia nigra and striatum in Parkinson's disease.

Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. These powerful techniques, when combined with data analysis tools, detect changes in transcript abundance in diseased tissue relative to control. We hypothesize that specific differential gene expression underlies important pathogenic processes in Parkinson's disease, which is characterized by the gradual loss of dopaminergic neurons in the substantia nigra and consequent loss of dopamine in the striatum. We have therefore examined gene expression levels in the human parkinsonian nigrostriatal pathway, and compared them with those of neurologically normal controls. Using unsupervised clustering methods, we demonstrate that relatively few genes' expression levels can effectively distinguish between disease and control brains. Further, we identify several interesting patterns of gene expression that illuminate pathogenic cascades in Parkinson's disease. In particular is the robust loss of synaptic gene expression in diseased substantia nigra and striatum.

Aged↗
PubMed2004

Rhythmic melatonin secretion does not correlate with the expression of arylalkylamine N-acetyltransferase, inducible cyclic amp early repressor, period1 or cryptochrome1 mRNA in the sheep pineal.

The pineal gland, through nocturnal melatonin, acts as a neuroendocrine transducer of daily and seasonal time. Melatonin synthesis is driven by rhythmic activation of the rate-limiting enzyme, arylalkylamine N-acetyltransferase (AA-NAT). In ungulates, AA-NAT mRNA is constitutively high throughout the 24-h cycle, and melatonin production is primarily controlled through effects on AA-NAT enzyme activity; this is in contrast to dominant transcriptional control in rodents. To determine whether there has been a selective loss of circadian control of AA-NAT mRNA expression in the sheep pineal, we measured the expression of other genes known to be rhythmic in rodents (inducible cAMP early repressor ICER, the circadian clock genes Period1 and Cryptochrome1, as well as AA-NAT). We first assayed gene expression in pineal glands collected from Soay sheep adapted to short days (Light: dark, 8-h: 16-h), and killed at 4-h intervals through 24-h. We found no evidence for rhythmic expression of ICER, AA-NAT or Cryptochrome1 under these conditions, whilst Period1 showed a low amplitude rhythm of expression, with higher values during the dark period. In a second group of animals, lights out was delayed by 8-h during the final 24-h sampling period, a manipulation that causes an immediate shortening of the period of melatonin secretion. This did not significantly affect the expression of ICER, AA-NAT or Cryptochrome1 in the pineal, whilst a slight suppressive effect on overall Per1 levels was observed. The attenuated response to photoperiod change appears to be specific to the ovine pineal, as the first long day induced rapid changes of Period1 and ICER expression in the hypothalamic suprachiasmatic nuclei and pituitary pars tuberalis, respectively. Overall, our data suggest a general reduction of circadian control of transcript abundance in the ovine pineal gland, consistent with a marked evolutionary divergence in the mechanism regulating melatonin production between terrestrial ruminants and fossorial rodents.

Animals↗
PubMed2026

Ramu stunt virus genome reveals previously unreported segments and nucleocapsid domain duplication in Mechlorovirus.

Ramu stunt virus (RmSV), a member of the genus Mechlorovirus within the family Phenuiviridae, was previously described as a six-segmented RNA virus infecting sugarcane. In this study, we re-examined type material and additional isolates using high-throughput sequencing and RT-PCR validation, revealing that RmSV possesses a nine-segmented genome, making it the largest reported in the Phenuiviridae. This expanded architecture includes duplicated RNA segments (RNA 2a and RNA 2b) encoding nucleocapsid-like proteins and two novel segments (RNA 7 and RNA 8). Comparative analysis showed that RNA 2a and 2b share about 84% amino acid identity, while RNA 5 encodes a third nucleocapsid homolog, indicating unprecedented domain redundancy. Structural modeling confirmed that all three nucleocapsid proteins maintain a conserved fold despite low sequence identity, with electrostatic mapping suggesting differential RNA-binding potential. Additionally, RNA 6 encodes a hypothetical protein structurally similar to the rice stripe virus disease-specific S-protein, implicating a role in symptom development. Transcript abundance analysis revealed RNA 6 as the most highly expressed segment across isolates. These findings revise the genomic composition of RmSV, highlight mechanisms of genome plasticity and adaptive evolution in plant-infecting bunyaviruses, and underscore practical implications for diagnostic assay design, resistance breeding, and biosecurity surveillance.

Genome, Viral↗
PubMed2026

Methane and carbon dioxide emissions from wastewater treatment units linked to DOM stabilization and phosphonate-scavenging microbiomes.

Municipal wastewater treatment plants (WWTPs) are major engineered facilities for urban carbon removal, yet methane (CH4) formation and source mechanisms in downstream stages after aeration and biological nutrient removal remain poorly resolved. Process resolved monitoring at a full-scale WWTP showed that CH4 emissions were concentrated upstream, while measurable fluxes persisted in downstream sedimentation and denitrification units. Dissolved CH4 profiles showed strong attenuation of influent derived CH4 during upstream treatment, followed by a local increase after secondary clarification. Carbon dioxide (CO2) emissions peaked in the biochemical tank, consistent with rapid oxidation of labile organic carbon. Fluorescence and molecular analyses revealed a shift in dissolved organic matter (DOM) from protein like to more humic and processed molecules, while community assembly remained predominantly deterministic despite greater stochasticity in later stages. The C-P lyase catalytic core was enriched in these units, accompanied by higher phnJ transcript abundance, candidate organophosphonate features, and genomic potential. Together with BES insensitive CH4 formation in independent microcosms, these observations supported C-P lyase mediated organophosphonate utilization as a contributing pathway to local CH4 formation. Integrated evidence indicated that DOM stabilization, deterministic community filtering, potential succinate mediated cross feeding, and phosphonate scavenging jointly shaped this process. These findings show that advanced treatment units are not CH4 hotspots, but neither are they CH4 inactive zones; process resolved GHG assessments should therefore consider persistent local CH4 generation and its association with substrate restructuring and alternative phosphorus acquisition.

Dissolved organic matter↗
PubMed2004

RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos.

Both murine endogenous retrovirus-L (MuERV-L) and intracisternal A particle (IAP), two autonomous long terminal repeat (LTR) retrotransposons, are activated during genome activation in the preimplantation mouse embryo, and both sense and antisense transcripts are detected in 2-cell and 8-cell stage embryos. Because RNA interference (RNAi) functions in the preimplantation mouse embryo, we analyzed the relationship between RNAi and MuERV-L and IAP expression by inhibiting RNAi and measuring relative changes of the levels of these transcripts. We inhibited the initial step in the RNAi pathway by injecting 1-cell embryos with mDicer siRNA or long mDicer dsRNA and analyzed MuERV-L and IAP expression at the 8-cell stage. This approach resulted in the targeted destruction of mDicer mRNA, but not Hdac1 mRNA, inhibited the RNAi pathway, and resulted in a 50% increase in IAP and MuERV-L transcript abundance. These results suggest that RNAi constrains expression of repetitive parasitic sequences in preimplantation embryos, and thereby contributes to preserving genomic integrity at a stage of development when the organism consists of only a few cells.

Animals↗
PubMed2006

Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling.

Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2004

Localization and expression of aromatase mRNA in adult zebrafish.

Estradiol plays a key role in the control of many behavioral and physiological aspects of reproduction therefore the expression of cytochrome P450 aromatase (CYP19), the enzyme responsible for the conversion of androgens to estrogens, is of vital interest. The zebrafish, and many other teleosts, have two aromatase genes (CYP19A1 and CYP19A2) that are expressed predominantly in the ovary and brain, respectively, however, the physiological impact of extra-gonadal aromatase has been poorly described. In this study, in situ hybridizations of whole-mount and paraffin sections of adult zebrafish brains, pituitaries, and ovarian follicles showed that CYP19A2 was strongly expressed in the olfactory bulb (OB), ventral telencephalon (TEL), preoptic area (POA), and ventral/caudal hypothalamic zone (HT) of the brain, and in the anterior and posterior lobes of the pituitary. The regional distribution of the CYP19A2 mRNA did not vary with sex however transcript abundance varied within (male "high expressers" had much higher expression in the OB, TEL, and HT than in "low expressers") and between sexes (higher in OB, TEL, and HT of males than in females). In situ hybridizations of CYP19A1 failed to develop a signal in the brain or pituitary but were detectable by RT-PCR. CYP19A1 was highly expressed in Stage III B follicles (>500 nm) with significantly lower levels in the Stage IV follicles (>680 nm), Stage III A follicles (>350 nm), and Stage I and II follicles (350 microm) which were embedded in connective tissues. The differential expression of the aromatase genes, particularly CYP19A2 in the brain, suggests that the two aromatase genes play different roles in the reproductive behavior and/or physiology of bony fish.

Animals↗
PubMed2006

Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice (Oryza sativa).

Small auxin-up RNAs (SAURs) are the early auxin-responsive genes represented by a large multigene family in plants. Here, we report the identification of 58 OsSAUR gene family members from rice (Oryza sativa japonica cv Nipponbare), the model monocot plant, by a reiterative database search and manual reannotation; 2 of these are pseudogenes. The coding sequences of OsSAURs do not possess any intron. Most of the predicted OsSAUR protein sequences harbor a putative nuclear localization signal at their N-terminus. Localized gene duplications appear to be the primary genetic event responsible for SAUR gene family expansion in rice. Interestingly, the duplication of OsSAURs was found to be associated with the chromosomal block duplication as well. The phylogenetic analysis revealed that the SAUR gene family expanded in rice and Arabidopsis due to species-specific expansion of the family in monocots and dicots. The auxin-responsive elements and downstream element are conserved in the upstream and downstream sequences, respectively, of OsSAURs. In addition to the 21 OsSAURs with full-length cDNA sequences and 20 with expressed sequence tags, gene expression analyses of at least 7 OsSAURs by RT-qPCR indicated that the majority of identified OsSAURs most likely are expressed in rice. The transcript abundance of the OsSAURs examined increased within a few minutes of exogenous auxin application with varying kinetics. The present study provides basic genomic information for the rice SAUR gene family and will pave the way for deciphering the precise role of SAURs in plant growth and development.

Base Sequence↗
PubMed1999

Early induction of angiogenetic signals in gliomas of GFAP-v-src transgenic mice.

Angiogenesis is a prerequisite for solid tumor growth. Glioblastoma multiforme, the most common malignant brain tumor, is characterized by extensive vascular proliferation. We previously showed that transgenic mice expressing a GFAP-v-src fusion gene in astrocytes develop low-grade astrocytomas that progressively evolve into hypervascularized glioblastomas. Here, we examined whether tumor progression triggers angiogenetic signals. We found abundant transcription of vascular endothelial growth factor (VEGF) in neoplastic astrocytes at surprisingly early stages of tumorigenesis. VEGF and v-src expression patterns were not identical, suggesting that VEGF activation was not only dependent on v-src. Late-stage gliomas showed perinecrotic VEGF up-regulation similarly to human glioblastoma. Expression patterns of the endothelial angiogenic receptors flt-1, flk-1, tie-1, and tie-2 were similar to those described in human gliomas, but flt-1 was expressed also in neoplastic astrocytes, suggesting an autocrine role in tumor growth. In crossbreeding experiments, hemizygous ablation of the tumor suppressor genes Rb and p53 had no significant effect on the expression of VEGF, flt-1, flk-1, tie-1, and tie-2. Therefore, expression of angiogenic signals is an early event during progression of GFAP-v-src tumors and precedes hypervascularization. Given the close similarities in the progression pattern between GFAP-v-src and human gliomas, the present results suggest that these mice may provide a useful tool for antiangiogenic therapy research.

Animals↗
PubMed1992

Expression, purification and biochemical comparison of natural and recombinant human non-pancreatic phospholipase A2.

The gene coding for human non-pancreatic phospholipase A2 (npPLA2) was cloned in a eukaryotic expression vector and transfected into chinese hamster ovary (CHO) cells. A number of cell lines stably expressing npPLA2 were obtained. Northern analysis of these cell lines showed an abundant transcript of expected size 1200 nt. The recombinant enzyme was efficiently secreted in quantities up to 400 micrograms npPLA2 per liter culture medium in the most productive cell lines. npPLA2 was purified to homogeneity from conditioned medium as previously described (1). The recombinant npPLA2 migrated by SDS--PAGE as a single band with an apparent mass of 14,000. The recombinant enzyme displayed the pH-optimum, calcium dependence and substrate preference that were characteristic of the human platelet and synovial fluid enzymes.

Animals↗