Biomedical subjects

R Reeves

Publications and source records attributed to R Reeves.

At least 19 recordsLinked to original sources

PubMed2004

In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice.

Transgenic mice expressing the luciferase (luc) gene under the control of the heme oxygenase-1 promoter (Ho1) were used to measure the induction of heme oxygenase in response to known toxicants. Transgenic Ho1-luc expression was visualized in vivo using a low-light imaging system (IVIS). Ho1-luc activation was compared to Ho1-luc expression, HO1 protein levels, standard markers of toxicity, and histology. Male and female Ho1-luc transgenic mice were exposed to acute doses of cadmium chloride (CdCl2, 3.7 mg/kg), doxorubicin (15 mg/kg), and thioacetamide (300 mg/kg). These agents induced the expression of Ho1-luc in the liver and other tissues to varying degrees. The greatest increase in Ho1-luc activity was observed in the liver in response to CdCl2; intermediate responses were observed for doxorubicin and thioacetamide. Induction of the Ho1-luc transgene by these agents was similar to endogenous protein levels of heme oxygenase as assessed by Western blotting, and generally correlated with plasma levels of circulating enzymes reflecting hepatic or general tissue damage. Histopathology confirmed the toxic effects of CdCl2 on liver and kidney; doxorubicin on kidney, liver, and intestine; and thioacetamide on the liver. Tissue damage was much more pronounced than the luciferase expression following thioacetamide treatment when compared with tissue damage and bioluminescence of the other toxicants. Nevertheless, the induction of Ho1-luc expression following exposure to these agents suggests that the Ho1-luc transgenic mouse may prove useful as a model for in vivo screening of compounds that induce luciferase expression as a marker of toxicity.

Alanine Transaminase↗

PubMed2004

Polarization observations with the Cosmic Background Imager.

Polarization observations of the cosmic microwave background with the Cosmic Background Imager from September 2002 to May 2004 provide a significant detection of the E-mode polarization and reveal an angular power spectrum of polarized emission showing peaks and valleys that are shifted in phase by half a cycle relative to those of the total intensity spectrum. This key agreement between the phase of the observed polarization spectrum and that predicted on the basis of the total intensity spectrum provides support for the standard model of cosmology, in which dark matter and dark energy are the dominant constituents, the geometry is close to flat, and primordial density fluctuations are predominantly adiabatic with a matter power spectrum commensurate with inflationary cosmological models.

Journal Article↗

PubMed2004

Expression of behavioral sensitization to the cocaine-like fungicide triadimefon is blocked by pretreatment with AMPA, NMDA and DA D1 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA), much like cocaine. A recent study in our laboratory found that intermittent injections of TDF led to robust locomotor sensitization in response to challenge TDF after a 2-week withdrawal period. The current study sought to determine whether the expression of TDF behavioral sensitization could be prevented by the DA D1-like receptor antagonist SCH 23390 (SCH), the DA D2-like receptor antagonist remoxipride (Rem), the competitive NMDA antagonist CPP, or the AMPA antagonist NBQX. Adult male C57/BL6 mice were injected with vehicle or 75 mg/kg TDF twice a week for 7 weeks, with locomotor activity measured periodically across the 14 doses. After a 2-week withdrawal period, mice were pretreated with SCH (0.015 mg/kg), Rem (0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg) followed 30 min later by vehicle or 75 mg/kg TDF and tested for the expression of TDF sensitization. Intermittent administration of TDF led to the development and robust expression of behavioral sensitization in terms of vertical activity. Pretreatment with SCH, NBQX and CPP successfully blocked the expression of vertical sensitization to TDF, while Rem pretreatment did not. All four antagonists, however, attenuated the neurochemical changes normally associated with TDF sensitization as measured 8 h after the 2-week TDF challenge. This paper reveals that NMDA, AMPA and DA D1-like receptors are necessary for the behavioral expression of sensitization to the fungicide triadimefon.

Animals↗

PubMed2004

Development of behavioral sensitization to the cocaine-like fungicide triadimefon is prevented by AMPA, NMDa, DA D1 but not DA D2 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA) and leads to increased locomotor activity levels in mice and rats, effects similar to those of indirect DA agonists such as cocaine. We recently found in mice that intermittent TDF administration led to robust locomotor sensitization, a phenomenon reflecting neuronal plasticity, following challenge with the same TDF dose after a 2-week withdrawal period. The current study sought to determine whether antagonists to DA D1-like receptors (SCH 23390; SCH), DA D2-like receptors (remoxipride; Rem), ionotropic glutamate n-methyl-d-aspartate (NMDA) receptors (CPP), or ionotropic glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NBQX) could prevent the development of TDF behavioral sensitization, therefore indicating their mechanistic involvement in TDF sensitization. Mice were treated with either vehicle, SCH (0.015 mg/kg), remoxipride (Rem, 0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg), followed 30 min later by vehicle or 75 mg/kg TDF (TDF), twice a week for 7 weeks, with locomotor activity measured post-dosing once a week. After a 2-week withdrawal period, mice were challenged with 75 mg/kg TDF or vehicle, to test for the presence of behavioral sensitization. Pretreatment with SCH, CPP, or NBQX, but not Rem, blocked the development of behavioral sensitization to TDF specifically for vertical activity. Antagonists that blocked TDF vertical sensitization also attenuated the increase in extracellular DA turnover (homovanillic acid [HVA]/DA) normally associated with this behavioral response. Therefore, DA D1, NMDA and AMPA receptors appear to be necessary for the development of behavioral sensitization to TDF. As such, TDF may be considered an environmental risk factor for behavioral dysfunctions linked to glutamatergic and dopaminergic systems.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2003

Interactions of paraquat and triadimefon: behavioral and neurochemical effects.

Triadimefon (TDF), a triazole fungicide, and paraquat (PQ), a non-selective herbicide/dessicant, are both known to adversely impact brain dopaminergic function and are used in overlapping geographical areas of the US. Since "real world" situations indicate humans are exposed to a diverse mixture of chemicals, this study hypothesized that combined exposures to PQ+TDF could produce interactive effects by simultaneously attacking multiple target sites of dopamine systems. Thus, 10 mg/kg PQ (PQ10) and 25 or 50 mg/kg TDF (TDF25 and 50, respectively) were administered i.p. to male C57BL/6 mice, 2x per week for 12 weeks, either alone or in combination. Acutely, TDF50 increased horizontal and vertical activity with increased vertical activity still occurring 24h later, indicative of sustained behavioral sensitization. Acutely, PQ decreased horizontal but not vertical activity with a lack of residual effects at 24h. PQ prevented the increased levels of activity associated with TDF50. These interactions differed for horizontal and vertical activity, indicating their differential neurochemical mediation, and suggesting that they did not arise from simple additivity of PQ and TDF effects. Nor could the interactive effects be readily ascribed to corresponding neurochemical interactions, since all treatments generally increased levels of DA and metabolites acutely in striatum and were associated with general reductions in levels of DA and metabolites and turnover in striatum and frontal cortex 7 days after the final treatment. Thus, TDF and PQ both separately and through interactions may serve as environmental risk factors through different mechanisms for dopaminergically-mediated behavioral dysfunctions.

Animals↗

PubMed2001

Molecular biology of HMGA proteins: hubs of nuclear function.

Members of the HMGA (a.k.a. HMGI/Y) family of 'high mobility group' (HMG) proteins participate in a wide variety of nuclear processes ranging from chromosome and chromatin mechanics to acting as architectural transcription factors that regulate the expression of numerous genes in vivo. As a consequence, they function in the cell as highly connected 'nodes' of protein-DNA and protein-protein interactions that influence a diverse array of normal biological processes including growth, proliferation, differentiation and death. The HMGA proteins, likewise, participate in pathological processes by, for example, acting as regulators of viral gene transcription and by serving as host-supplied proteins that facilitate retroviral integration. HMGA genes are bona fide proto-oncogenes that promote tumor progression and metastasis when overexpressed in cells. High constitutive HMGA protein levels are among the most consistent feature observed in all types of cancers with increasing concentrations being correlated with increasing malignancy. The intrinsic attributes that endow the HMGA proteins with these remarkable abilities are a combination of structural, biochemical and biological characteristics that are unique to these proteins. HMGA proteins have little, if any, secondary structure while free in solution but undergo disordered-to-ordered structural transitions when bound to substrates such as DNA or other proteins. Each protein contains three copies of a conserved DNA-binding peptide motif called the 'AT-hook' that preferentially binds to the minor groove of stretches of AT-rich sequence. In vivo HMGA proteins specifically interact with a large number of other proteins, most of which are transcription factors. They are also subject to many types of in vivo biochemical modifications that markedly influence their ability to interact with DNA substrates, other proteins and chromatin. And, most importantly, both the transcription of HMGA genes and the biochemical modifications of HMGA proteins are direct downstream targets of numerous signal transduction pathways making them exquisitely responsive to various environmental influences. This review covers recent advances that have contributed to our understanding of how this constellation of structural and biological features allows the HMGA proteins to serve as central 'hubs' of nuclear function.

Amino Acid Sequence↗

PubMed2001

Sequence and analysis of the murine Hmgiy (Hmga1) gene locus.

The HMGIY non-histone proteins play important roles as architectural transcription factors that regulate gene transcription in mammalian cells and also act as host-supplied cofactors necessary for retroviral integration. The genes coding for the HMGIY proteins are proto-oncogenes, and their aberrant or over-expression is correlated with both neoplastic transformation and metastatic progression in a wide variety of tumors. Here, we report the first complete sequence of the murine Hmgiy (a.k.a. Hmga1) gene and provide a detailed comparison of this with the sequence and organization of the human HMGIY gene, including an analysis of its promoter region with the previously unreported 5' upstream region of the human gene. These analyses reveal a remarkable degree of overall sequence conservation in both the protein coding and promoter regions of the murine and human genes, including conservation of the c-Myc binding site that has been demonstrated to regulate murine Hmgiy transcription (Wood et al., 2000. Mol. Cell. Biol. 20, 5490-5502). The promoters of both genes contain other conserved transcription factor binding sites that may also represent important cis-regulatory elements. Two exons present in the 5' untranslated region of the human gene, however, are missing from the murine gene, suggesting that these two closely related mammalian species regulate transcription of their Hmgiy genes in an individualistic manner.

Animals↗

PubMed2001

HMGI/Y proteins: flexible regulators of transcription and chromatin structure.

The mammalian HMGI/Y (HMGA) non-histone proteins participate in a wide variety of cellular processes including regulation of inducible gene transcription, integration of retroviruses into chromosomes and the induction of neoplastic transformation and promotion of metastatic progression of cancer cells. Recent advances have contributed greatly to our understanding of how the HMGI/Y proteins participate in the molecular mechanisms underlying these biological events. All members of the HMGI/Y family of 'high mobility group' proteins are characterized by the presence of multiple copies of a conserved DNA-binding peptide motif called the 'AT hook' that preferentially binds to the narrow minor groove of stretches of AT-rich sequence. The mammalian HMGI/Y proteins have little, if any, secondary structure in solution but assume distinct conformations when bound to substrates such as DNA or other proteins. Their intrinsic flexibility allows the HMGI/Y proteins to participate in specific protein-DNA and protein-protein interactions that induce both structural changes in chromatin substrates and the formation of stereospecific complexes called 'enhanceosomes' on the promoter/enhancer regions of genes whose transcription they regulate. The formation of such regulatory complexes is characterized by reciprocal inductions of conformational changes in both the HMGI/Y proteins themselves and in their interacting substrates. It may well be that the inherent flexibility of the HMGI/Y proteins, combined with their ability to undergo reversible disordered-to-ordered structural transitions, has been a significant factor in the evolutionary selection of these proteins for their functional role(s) in cells.

Amino Acid Sequence↗

PubMed2001

Effects of cyclosporin A administered into the airways against antigen-induced airway inflammation and hyperreactivity in the rat.

The immunosuppressant cyclosporin A given orally has anti-asthma properties but carries an undesirable risk of systemic effects. We administered cyclosporin A to Brown Norway rats either orally (p.o.) or topically by intratracheal (i.t.) instillation into the airways before inhaled antigen. Cyclosporin A suppressed the antigen-induced accumulation of activated (CD25+) CD4+ T lymphocytes and eosinophils in the lung, interleukin-5 mRNA expression in lung tissue and airway hyperreactivity. Intratracheal cyclosporin A suppressed cell accumulation at a 10-fold lower dose than that required orally. Minimum effective doses were 3 mg x kg(-1) i.t. and 30 mg x kg(-1) p.o. Intratracheal administration reduced the plasma concentration and systemic exposure compared with an equieffective oral dose, but the reduction (4-5-fold) was not as large as anticipated. Our data suggests that although topical administration to asthmatics would provide some potential for an improved safety margin, it may not offer any major advantage over existing oral therapy. However, the data clearly demonstrate that a novel immunosuppressant with similar anti-inflammatory properties but reduced potential for systemic effects would offer an attractive therapy for severe asthma.

Administration, Inhalation↗

PubMed2001

Down-regulation of high mobility group-I(Y) protein contributes to the inhibition of nitric-oxide synthase 2 by transforming growth factor-beta1.

The inducible isoform of nitric-oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), which participates in the pathophysiology of systemic inflammatory diseases such as sepsis. NOS2 is transcriptionally up-regulated by endotoxin and inflammatory cytokines, and down-regulated by transforming growth factor (TGF)-beta1. Recently we have shown that high mobility group (HMG)-I(Y) protein, an architectural transcription factor, contributes to NOS2 gene transactivation by inflammatory mediators. The aim of the present study was to determine whether regulation of HMG-I(Y) by TGF-beta1 contributes to the TGF-beta1-mediated suppression of NOS2. By Northern blot analysis, we show that TGF-beta1 decreased cytokine-induced HMG-I(Y) mRNA levels in vascular smooth muscle cells and macrophages in vitro and in vivo. Western analysis confirmed the down-regulation of HMG-I(Y) protein by TGF-beta1. To determine whether the down-regulation of HMG-I(Y) contributed to a decrease in NOS2 gene transactivation by TGF-beta1, we performed cotransfection experiments. Overexpression of HMG-I(Y) was able to restore cytokine inducibility of the NOS2 promoter that was suppressed by TGF-beta1. The effect of TGF-beta1 on NOS2 gene transactivation was not related to a decrease in binding of HMG-I(Y) to the promoter of the NOS2 gene, but due to a decrease in endogenous HMG-I(Y) protein. These data provide the first evidence that cytokine-induced HMG-I(Y) can be down-regulated by TGF-beta1. This down-regulation of HMG-I(Y) contributes to the TGF-beta1-mediated decrease in NOS2 gene transactivation by proinflammatory stimuli.

Animals↗

PubMed2001

Intrinsically disordered protein.

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Models, Molecular↗

PubMed2001

Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro.

We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.

Avian Sarcoma Viruses↗

PubMed2001

Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells.

Numerous studies have demonstrated that overexpression or aberrant expression of the HMGI(Y) family of architectural transcription factors is frequently associated with both neoplastic transformation of cells and metastatic tumor progression. Little is known, however, about the molecular roles played by the HMGI(Y) proteins in these events. Here we report that human breast epithelial cells harboring tetracycline-regulated HMGI(Y) transgenes acquire the ability to form both primary and metastatic tumors in nude mice only when the transgenes are actively expressed. Unexpectedly, the HMG-Y, rather than the HMG-I, isoform of these proteins is the most effective elicitor of both neoplastic transformation and metastatic progression in vivo. Furthermore, expression of either antisense or dominant-negative HMGI(Y) constructs inhibits both the rate of proliferation of tumor cells and their ability to grow anchorage independently in soft agar. Array analysis of transcription profiles demonstrates that the HMG-I and HMG-Y isoform proteins each modulate the expression of distinctive constellations of genes known to be involved in signal transduction, cell proliferation, tumor initiation, invasion, migration, induction of angiogenesis, and colonization. Immunohistochemical analyses of tumors formed in nude mice indicate that many have undergone an epithelial-mesenchymal transition in vivo. Together, these findings demonstrate that overexpression of the HMGI(Y) proteins, more specifically, the HMG-Y isoform protein, is causally associated with both neoplastic transformation and metastatic progression and suggest that induction of integrins and their signaling pathways may play significant molecular roles in these biological events.

Animals↗

PubMed2000

Differential in vivo modifications of the HMGI(Y) nonhistone chromatin proteins modulate nucleosome and DNA interactions.

The HMGI(Y) family of "high mobility group" nonhistone proteins are architectural transcription factors whose overexpression is highly correlated with both cancerous transformation and increased malignancy and metastatic potential of tumors in vivo. Here we report on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. The MCF-7 cell line exhibits many characteristics of normal breast epithelial cells and does not form tumors when injected into nude mice, whereas the MCF-7/PKC-alpha cell line, a derivative of MCF-7 that expresses a transgene coding for the enzyme protein kinase C-alpha (PKC-alpha), is both malignant and highly metastatic. Using MALDI mass spectrometry, we show that the HMG-Y protein is more highly modified than the HMG-I protein in both the MCF-7 and the MCF-7/PKC-alpha cells. Significantly, the HMG-Y protein isolated from the highly metastatic MCF-7/PKC-alpha cells possesses a unique constellation of phosphorylations, methylations, and acetylations not found on the HMG-I protein isolated from either the MCF-7 or MCF-7/PKC-alpha cells. We further demonstrate that some of the same amino acid residues phosphorylated on recombinant HMGI(Y) proteins by purified PKC in vitro are also phosphorylated on the HMG-I(Y) proteins isolated from MCF-7/PKC-alpha cells, suggesting that PKC phosphorylates these proteins in vivo. Quantitative substrate binding analyses indicate that the biochemical modifications present on the HMG-I and HMG-Y proteins differentially influence the ability of these proteins to interact with both A.T-rich DNA substrates and nucleosome core particles in vitro, suggesting a similar modulation of such binding affinities in vivo. To our knowledge, this is the first demonstration of differences in the types of in vivo biochemical modifications found on the HMG-I and HMG-Y proteins in cells and also the first experimental evidence suggesting a possible linkage between such posttranslational modifications and the neoplastic potential of cells.

AT Rich Sequence↗

PubMed2000

Headache in mild-to-moderate hypertension and its reduction by irbesartan therapy.

BACKGROUND: Although it is generally acknowledged to be a problem in severe hypertension, headache has not been consistently associated with mild-to-moderate hypertension. PATIENTS AND METHODS: In 7 randomized, double-blind, placebo-controlled trials, which included 2,673 patients with mild-to-moderate hypertension (defined as seated diastolic blood pressure of 95-110 mm Hg), patients were randomized to receive once-daily treatment with irbesartan, an angiotensin II receptor blocker (n= 1,987), or placebo (n=686). The data were pooled and analyzed retrospectively to determine whether the level of hypertension was associated with headache and whether antihypertensive therapy reduced the incidence of headache. RESULTS: Factors found to be predictive of headache incidence were diastolic blood pressure, sex (female), and age (<50 years). In comparison with placebo, the use of irbesartan was associated with a significant reduction in the incidence of headache (P=.003). CONCLUSIONS: These data suggest that mild-to-moderate hypertension is not asymptomatic and that the incidence of headache can be reduced by antihypertensive treatment with a favorable adverse effect profile.

Adult↗

PubMed2000

The role of high-mobility group I(Y) proteins in expression of IL-2 and T cell proliferation.

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-kappaB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.

Base Sequence↗

PubMed2000

XREFdb: cross-referencing the genetics and genes of mammals and model organisms.

XREFdb supports the investigation of protein function in the context of information available through work in multiple organisms. In addition to facilitating the association of functional data among known genes from multiple organisms, XREFdb has developed strategies that provide access to information associated with as-yet unstudied genes. The database organizes protein similarity and genetic map positional information from diverse sources in the public domain to facilitate investigator evaluation of potential functional significance. XREFdb is found at URL www.ncbi.nlm.nih.gov/XREFdb

Animals↗

PubMed2000

Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.

Blotting, Northern↗