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Biomedical subjects

R Reeves

Publications and source records attributed to R Reeves.

At least 55 records · Page 3Linked to original sources
PubMed1998

Tumor promoter induces high mobility group HMG-Y protein expression in transformation-sensitive but not -resistant cells.

Elevated levels of high mobility group (HMG) nonhistone chromosomal proteins I and Y, alternatively spliced members of the HMG-I(Y) family of architectural transcription factors, have been linked with human cancer and with neo-plastic and metastatic phenotypes in model systems. To investigate whether HMG-I(Y) proteins may influence susceptibility to neoplastic transformation, HMG-I(Y) mRNA and protein levels were compared in the JB6 murine model of neoplastic progression. HMG-I(Y) mRNAs were expressed at very low levels in preneoplastic, transformation-resistant (P-) cell lines and were constitutively expressed at much higher levels in both transformation-sensitive (P +) and transformed (Tx) tumorigenic cell lines. HMG-I(Y) mRNAs were induced to higher levels by the tumor promoter 12-O-tetradecanoylphorbol acetate (TPA) and were sustained longer in P+ than in P- cells. Nevertheless, in both P- and P+ cells, primer extension analysis revealed that the same four major HMG-I(Y) gene transcription start sites were utilized with or without TPA treatment. RT-PCR revealed that there was always slightly more Y than I form mRNA present in all of the variant JB6 cell lines. Immunoblotting indicated that both HMG-I and -Y proteins increased in P + cells in response to TPA treatment. Remarkably, in P- cells treated with TPA, only HMG-I (and not HMG-Y) protein levels increased. This unique differential TPA-induction of the HMG-Y protein in JB6 variants suggests a role for HMG-Y in mediating tumor promoter-induced neoplastic transformation. Furthermore, these results demonstrate that HMG-I and Y protein translation and/or stability is differently regulated in JB6 P- cells and provide the first indication that I and Y proteins may have different functions.

Alternative Splicing↗
PubMed1998

Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions.

Known transcription factor-DNA interactions in the minimal enhancer of the murine interleukin-2 gene (IL-2) do not easily explain the T cell specificity of IL-2 regulation. To seek additional determinants of cell type specificity, in vivo methodologies were employed to examine chromatin structure 5' and 3' of the 300 bp IL-2 proximal promoter/enhancer region. Restriction enzyme accessibility revealed that until stimulation the IL-2 proximal promoter/enhancer exists in a closed conformation in resting T and non-T cells alike. Within this promoter region, DMS and DNase I genomic footprinting also showed no tissue-specific differences prior to stimulation. However, DNase I footprinting of the distal -600 to -300 bp region revealed multiple tissue-specific and stimulation-independent DNase I hypersensitive sites. Gel shift assays detected T cell-specific complexes binding within this region, which include TCF/LEF or HMG family and probable Oct family components. Upon stimulation, new DNase I hypersensitive sites appeared in both the proximal and distal enhancer regions, implying that there may be a functional interaction between these two domains. These studies indicate that a region outside the established IL-2 minimal enhancer may serve as a stable nucleation site for tissue-specific factors and as a potential initiation site for activation-dependent chromatin remodeling.

Animals↗
PubMed1998

Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture.

Biologically active human interleukin-2 (IL-2) and IL-4, key lymphokines involved in immune regulation, were produced and secreted into the medium by genetically modified Nicotiana tabacum cells grown in suspension culture. Secretion through the plasma membrane and cell wall into the medium was facilitated by the natural mammalian leader sequences. IL-2 and IL-4 were detected in the medium at concentrations of 0.10 and 0.18 microgram/mL, respectively, although higher levels were detected within the lymphokine-producing cells (approximately 0.80 microgram/mL for IL-2 and approximately 0.28 microgram/mL for IL-4). By Western blot, IL-4 was found to be secreted as two small polypeptides with molecular masses of approximately 18-20 kDa. The biological activity of IL-2 was determined by cell proliferation of the IL-2-dependent murine CTLL-2 cell line, while that of IL-4 was determined by cell proliferation of the CTLL-2 cell line [CT.h4S] which was stably transfected with the human IL-4 receptor. These findings indicate that plant suspension culture can be used to produce and secrete into the medium a variety of biologically active mammalian proteins that are of clinical and diagnostic relevance.

Base Sequence↗
PubMed1998

Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein.

MRC OX-2 is a rat type I membrane glycoprotein and a member of the immunoglobulin gene superfamily that has recently been shown to be able to costimulate murine T cell proliferation (Borriello et al. J. Immunol, 158, 4548, 1997). We now report the genomic organization for the gene encoding the murine homolog of MRC OX-2 (Mox2). The gene is composed of 6 exons and 5 introns spanning a minimum of 13.7 kb. Exon 1 encodes the amino terminal four amino acids and is located in the center of a 500-bp CpG island, suggestive of the presence of a promoter. Analysis of the sequences immediately upstream of exon 1, however, do not reveal a TATA or CAAT box. We also demonstrate that in addition to the canonical transcript, composed of exons 1 through 6, this gene gives rise to an additional nonproductive transcript resulting from the absence of exon 2, which leads to a frameshift and premature translation termination. The ratio of these alternative transcripts is not regulated by mitogenic stimulation with ConA or LPS. The Mox2 gene maps to Chr 16, telomeric to the clustered T cell costimulatory molecules Cd80 and Cd86 (Reeves et al. Genomics in press).

Amino Acid Sequence↗
PubMed1998

Spontaneous versus extraction related injuries associated with Accufix J-wire atrial pacemaker lead: tracking changes in patient management.

UNLABELLED: To make recommendations for management of potentially fatal failure of the Accufix series of atrial J-wire permanent pacemaker leads, we closely monitored the number of injuries and fatalities resulting either from spontaneous fracture of the J-wire or from attempts to extract the lead. In a population of 30,357 patients, 2,298 patients are enrolled in a prospective follow-up Multicenter Study, the remainder are patients with known clinical status from voluntary reporting, and 2,992 patients died following implant. In the remaining 27,365 patients, 6 deaths have been attributed to J-wire related injury (J-inj) while 13 were complications (E-inj) associated with 4,076 lead extraction procedures (3,974 intravascular (intra)/102 primary thoracotomy (PT). The date of occurrences were from 1994 to November 1997. CONCLUSIONS: (1) Since lead extractions were not conducted in a controlled study, it is not known whether the deaths associated with lead extraction is in excess of what would have occurred if these leads had not been removed in this specific subset. (2) Awareness of the procedure related complication rate appears to have moderated the rate of lead extraction and may ultimately lead to management that reasonably balances the risks of patient injury.

Electrodes, Implanted↗
PubMed1998

Kaplan-Meier analysis of freedom from extraction or death in patients with an Accufix J retention wire atrial permanent pacemaker lead: a potential management tool.

UNLABELLED: Morbidity (36 cases) and mortality (6 cases) have been reported in patients with Accufix J retention wire atrial leads. This has resulted in ongoing patient fluoroscopic monitoring as well as lead extractions. The estimated implanted worldwide population is 40,860. Estimating the size of the remaining population at risk is an important tool for assessing patient management guidelines. RESULTS: The Kaplan-Meier method can be used to calculate the cumulative probability of remaining free of extraction and death for patients based on implant duration. The individual Kaplan-Meier curves for lead extraction and patient survival can also be computed. Based on the Multicenter Study (MCS) population of 2,298 patients, the probability that a patient is alive with the lead still implanted at 5 years implant duration is 52.5%. The event-free survival rate at 5 years implant duration is 81.3%. The corresponding probability of remaining free from injury due to the J-wire is 99.9% at 5 years implant duration. Assuming similar rates of death and extraction, these results can be extrapolated to the world wide population. CONCLUSIONS: The management of Accufix patients must consider patient longevity, the probability of J-wire morbidity/mortality, and the probability of extraction complication morbidity/mortality. The probability of remaining at risk as a function of time from implant can be calculated from the events known in the MCS patient population. These event-free survival estimates can be used to identify subsets of the population at greater or lesser risk based on various clinical parameters.

Disease-Free Survival↗
PubMed1998

Cumulative hazard analysis of J-wire fracture in the Accufix series of atrial permanent pacemaker leads.

UNLABELLED: To permit a more complete analysis of J-wire fracture in the Accufix series of atrial permanent pacemaker leads, the time to occurrence of all known fractures and injuries has been redefined relative to the duration of risk exposure, that is, according to the interval of time between implant and occurrence of the event. This redefinition permits application of a cumulative hazards model to the data, which previously has not been explored. Predictors of J-wire fracture can be tested using this method. This also permits parametric curve-fitting for determination of linearity or constancy of risk of events over time. RESULTS: Among 2,063 Multicenter Study (MCS) leads analyzed, 381 fractures of the J-wire were identified. Stratified analysis based on cumulative hazard curves identified a more open shape of the J-wire as predictive of fracture, which supports the results previously reported based on logistic regression analysis. Fitting a Weibull curve to the cumulative hazard of J-wire fracture gives a shape parameter equal to 0.85. This value indicates that the instantaneous hazard of J-wire fracture decreased over time from implant. CONCLUSIONS: (1) The cumulative hazard function can be used to examine predictors of J-wire fracture and preliminary findings support the previously identified predictor of J shape; (2) Based on these analyses, the rate of J-wire fracture appears to decrease slightly as time from implant increases.

Electrodes, Implanted↗
PubMed1997

Silencer activity in the interferon-A gene promoters.

Interferon-A (IFN-A) differential gene expression is modulated by a complex interplay between cis-acting DNA elements and the corresponding specific trans-regulating factors. Substitutions in the proximal virus-responsive element of the interferon-A (IFN-A) promoters contribute to their differential gene expression. The 5' distal silencing region in the weakly virus-inducible murine IFN-A11 gene has been previously delimited. DNase I footprinting experiments and transient gene expression assays demonstrate identical silencing activity in equivalent regions of the genes for IFN-A11 and IFN-A4 promoters. A minimal 20-mer distal negative regulatory element (DNRE) in both promoters is necessary and sufficient for the silencing and a region in the highly inducible IFN-A4 promoter located between the silencer and the virus-responsive element overrides the silencer activity. Mutations in the central region of the DNRE, causing derepression, also altered the formation of one of the two major DNA-protein complexes. One of these contains a protein related to or identical to the high mobility group I(Y) proteins, while the other complex contains a major protein present in uninduced and virus-induced cells with a molecular mass of 38 kDa, which may be related to the silencer activity. Similar DNREs are present in other virus-uninducible IFN-A promoters, and these data suggest that a common silencer may mediate the transcriptional repression in different genes of this family.

Base Sequence↗
PubMed1997

Competition between HMG-I(Y), HMG-1 and histone H1 on four-way junction DNA.

High mobility group proteins HMG-I(Y) and HMG-1, as well as histone H1, all share the common property of binding to four-way junction DNA (4H), a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events. The structure of 4H has also been hypothesized to mimic the DNA crossovers occurring at, or near, the entrance and exit sites on the nucleosome. Furthermore, upon binding to either duplex DNA or chromatin, all three of these nuclear proteins share the ability to significantly alter the structure of bound substrates. In order to further elucidate their substrate binding abilities, electrophoretic mobility shift assays were employed to investigate the relative binding capabilities of HMG-I(Y), HMG-1 and H1 to 4H in vitro. Data indicate a definite hierarchy of binding preference by these proteins for 4H, with HMG-I(Y) having the highest affinity (Kd approximately 6.5 nM) when compared with either H1 (Kd approximately 16 nM) or HMG-1 (Kd approximately 80 nM). Competition/titration assays demonstrated that all three proteins bind most tightly to the same site on 4H. Hydroxyl radical footprinting identified the strongest site for binding of HMG-I(Y), and presumably for the other proteins as well, to be at the center of 4H. Together these in vitro results demonstrate that HMG-I(Y) and H1 are co-dominant over HMG-1 for binding to the central crossover region of 4H and suggest that in vivo both of these proteins may exert a dominant effect over HMG-1 in recognizing and binding to altered DNA structures, such as Holliday junctions, that have conformations similar to 4H.

Base Composition↗
PubMed1997

Genome cross-referencing and XREFdb: implications for the identification and analysis of genes mutated in human disease.

Comparative genomics approaches and multi-organismal biology are valuable tools for genetic analysis. Cross-species connections between genes mutated in human disease states and homologues in model organisms can be particularly powerful, as model-organism gene function data and experimental approaches can shed light on the molecular mechanisms defective in the disease. We describe a project that is systematically identifying novel expressed sequence tag (EST) sequences that are highly related to genes in model organisms and mapping them to positions on the mouse and human maps. This process effectively cross-references model organism genes with mapped mammalian phenotypes, facilitating the identification of genes mutated in human disease states via the positional candidate approach. A public database, XREFdb (http:@www.ncbi.nlm.nih.gov/XREFdb/), disseminates similarity search, mapping and mammalian phenotype information and increases the rate at which these cross-species connections are established.

Animals↗
PubMed1997

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

Amino Acid Sequence↗
PubMed1997

Effects of epidermal growth factor and estrogen on the regulation of the HMG-I/Y gene in human mammary epithelial cell lines.

Members of the HMG-I/Y family of high-mobility-group chromatin proteins have been demonstrated to regulate gene expression in human cells in vivo. They are thought to function as gene regulatory molecules by acting as architectural transcription factors that modulate DNA and/or chromatin structure. Numerous studies have indicated that elevated HMG-I/Y gene expression is directly correlated with more advanced cancers and with increased metastatic potential. The inducible expression of the HMG-I/Y gene was studied in two human mammary epithelial cell lines, MCF7 and Hs578T, in the presence, or absence, of either 17 beta-estradiol or epidermal growth factor (EGF). Northern blot analysis indicated that there was no increase in HMG-I/Y mRNA in the nonmetastatic MCF7 cells when they were treated with either 17 beta-estradiol or EGF. In contrast, in the highly metastatic Hs578T cell line, there is a dramatic induction of HMG-I/Y mRNA expression of up to 23-fold when the cells are treated with EGF. mRNA primer extension analysis indicated that only two (of the possible four different) transcription initiation start sites in the HMG-I/Y gene are induced by EGF treatment of the Hs578T cells. Additional experiments demonstrated that in both epithelial cell types HMG-I/Y mRNAs are very stable (tl/2 of approximately 30 hr) and that in the Hs578T cells treated with EGF the cellular concentrations of the HMG-I/Y proteins increase concurrently with the induced mRNA levels. Given that HMG-I/Y proteins are regulators of gene activity whose elevated in vivo concentrations are known to be correlated with increased metastatic potential, these data demonstrating an EGF-induced over-expression of HMG-I/Y in the highly metastatic Hs578T, but not in the nonmetastatic MCF7cells, may have important implications concerning the cellular mechanisms involved in the progression of mammary epithelial tumors.

Breast Neoplasms↗
PubMed1997

A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region.

DGS and VCFS, haploinsufficiencies characterized by multiple craniofacial and cardiac abnormalities, are associated with a microdeletion of chromosome 22q11.2. Here we document synteny between a 150-kb region on mouse chromosome 16 and the most commonly deleted portion of 22q11.2. Seven genes, all of which are transcribed in the early mouse embryo, have been identified. Of particular interest are two serine/threonine kinase genes and a novel goosecoid-like homeobox gene (Gscl). Comparative sequence analysis of a 38-kb segment reveals similarities in gene content, order, exon composition, and transcriptional direction. Therefore, if deletion of these genes results in DGS/VCFS in humans, then haploinsufficiencies involving this region of chromosome 16 should recapitulate the developmental field defects characteristic of this syndrome.

Abnormalities, Multiple↗
PubMed1997

Use of Iliad to improve diagnostic performance of nurse practitioner students.

Nurse practitioners (NPs) have dual goals as primary care providers, combining the traditional goals of nursing with extended goals as diagnosticians. Diagnostic reasoning, therefore, is a critical component of NP education. Iliad, a computerized diagnostic reasoning expert system, has been used effectively to teach diagnostic skills to medical students. A pilot study was undertaken to determine the effects of Iliad training on NP students' diagnostic skill performance and to identify technical and instructional issues of implementation. The study found that the use of Iliad improved NP students' diagnostic reasoning, and that the training effects were modified by prior nursing experience. Successful use of Iliad required planning, faculty commitment, and technical support.

Clinical Competence↗
PubMed1997

HMGI(Y) activation by chromosome 6p21 rearrangements in multilineage mesenchymal cells from pulmonary hamartoma.

The chromosome band 6p21 high-mobility group gene, HMGI(Y), encodes DNA-binding proteins with both chromatin structural and gene regulatory roles. Although HMGI(Y) expression is associated with neoplastic transformation, no oncogenic HMGI(Y) mutations have been identified. We report pulmonary chondroid hamartoma chromosome 6p21 aberrations targeting HMGI(Y). Several pulmonary chondroid hamartomas had chromosome rearrangements mapping within 100 kb of HMGI(Y), and one pulmonary chondroid hamartoma contained an intragenic fusion juxtaposing HMGI(Y) A.T book DNA-binding domains with a laminin alpha 4 chain epidermal-growth-factor-like/ zinc-finger-like motif. These findings demonstrate an oncogenic role for HMGI(Y) and for laminin chain transcriptional regulatory motifs.

Amino Acid Sequence↗
PubMed1997

Pim-1 protein expression is regulated by its 5'-untranslated region and translation initiation factor elF-4E.

Expression of Pim-1, an oncogenic serine/threonine kinase, is highly regulated at the transcriptional, posttranscriptional, and posttranslational levels. Here, we report that expression of Pim-1 kinase is additionally regulated at the translational level. Pim-1 protein expression did not increase in Hut-78 lymphocytes in response to PMA1/ionomycin stimulation despite approximately 20-fold increases in mRNA levels, suggesting that translation was repressed. Sequence analysis of the 5'-untranslated region (UTR) indicated a long (400 nucleotide), 76% G + C-rich region, characteristics known to inhibit translation. Deletion of the 5'-UTR of pim-1 increased translation of the Pim-1 protein approximately 10-fold in vitro in reticulocyte lysates and approximately 1.6-fold in vivo in NIH-3T3 cells. When full-length 5'-UTR-containing pim-1 cDNA constructs were transfected into NIH-3T3 cells overexpressing eukaryotic translation initiation factor 4E (eIF-4E), approximately 6-fold higher levels of Pim-1 protein were produced, as compared to that produced in control NIH-3T3 cells. Moreover, eIF-4E overexpression had little effect in the absence of the 5'-UTR, suggesting that it relieved 5'-UTR-mediated inhibition of Pim-1 expression.

3T3 Cells↗