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R C Nichols

Publications and source records attributed to R C Nichols.

At least 19 recordsLinked to original sources
PubMed2005

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Radiation therapy and concomitant paclitaxel/carboplatin chemotherapy for muscle invasive transitional cell carcinoma of the bladder: a well-tolerated combination.

This review evaluates tolerance and disease control for eight patients with muscle invasive bladder cancer treated with pelvic radiotherapy and concomitant paclitaxel/carboplatin chemotherapy. From October 1996 through February 1998, eight patients were treated with pelvic radiotherapy and concomitant paclitaxel/carboplatin chemotherapy. All received from 39.60-41.40 Gy to the pelvis followed by a boost to the initial site of disease. Final tumor doses ranged from 64.80-68.40 Gy. Most patients received paclitaxel at 150 mg/m2 and carboplatin at an area under the curve (AUC) of 7 at 3-week cycles during the radiation therapy. No patient required treatment interruption. With a median follow-up of 27 months, three patients remain free of local and distant disease at follow-up intervals of 24, 25, and 31 months. No surviving locally controlled patient demonstrated late urinary or gastrointestinal morbidity. All patients with a visibly complete transurethral resection of bladder tumor (TURBT) prior to radiotherapy achieved local disease control. For this group of patients, the absolute 2-year pelvic tumor control rate is 57%. The 2-year disease-specific survival is 43%. Paclitaxel/carboplatin chemotherapy can be delivered with continuous course pelvic radiation therapy without severe acute or apparent late toxicity. This combination also appears to be effective in achieving disease control in the urinary bladder, particularly in those patients who have undergone a thorough TURBT. The authors believe that it would be reasonable to investigate this combination in future bladder conservation protocols. The combination of paclitaxel and carboplatin with radiotherapy may be of particular value in elderly patients or those with renal impairment.

Aged↗
PubMed2000

Transcriptional regulation of the human acid alpha-glucosidase gene. Identification of a repressor element and its transcription factors Hes-1 and YY1.

Acid alpha-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid alpha-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5'-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TK promoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairy and Enhancer of split (Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid alpha-glucosidase gene as well as other lysosomal enzyme genes.

Base Sequence↗
PubMed2000

The RGG domain in hnRNP A2 affects subcellular localization.

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.

3T3 Cells↗
PubMed1999

hnRNP A2 and hnRNP L bind the 3'UTR of glucose transporter 1 mRNA and exist as a complex in vivo.

Recent work identified an RNA binding protein whose presence in brain tumors correlated with translational repression of Glut1 expression. RNase T1 mapping demonstrated that this protein bound an AU-rich response element (AURE) in the Glut1 3'UTR. Facilitated by its differential expression in brain tumor cytosols, we identified this Glut1 RNA binding protein as hnRNP A2. Studies further demonstrated that hnRNP A2 was the major Glut1 RNA binding activity in other cell lines. Recombinant hnRNP A2 exhibited equivalent Glut1 RNA binding specificity, quite distinct from the related AURE binding protein hnRNP A1. These data indicate that hnRNP A2 is the Glut1 AURE binding protein whose cytoplasmic expression in gliomas is associated with translational repression and mRNA instability. Using this approach, we also identified the other major Glut1 3'UTR RNA binding activity as hnRNP L. Stimuli (hypoxia and hypoglycemia) which increase Glut1 mRNA stability selectively decreased polysomal levels of hnRNP A2 and L. Immunoprecipitation demonstrated that hnRNP A2 and L exist as a complex in vivo. As a result of these and other studies, we conclude that hnRNP A2 and L associate in vivo and independently bind the 3'UTR of Glut1 mRNA.

3' Untranslated Regions↗
PubMed1997

Modulation of AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes. The roles of cytoplasmic location, transcription, and phosphorylation.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A)+ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.

Adenosine↗
PubMed1996

A model of mRNA splicing in adult lysosomal storage disease (glycogenosis type II).

Glycogenosis type II is a recessively inherited disorder caused by mutations in the acid maltase (GAA) gene. Clinically, three different phenotypes are recognized: Infantile, juvenile and adult forms. A majority of compound heterozygous adult-onset patients carry a t-13g mutation in intron 1 associated with splicing out the first coding exon (exon 2). We have studied the mechanism of this mutation in a model system with wild-type and mutant minigenes expressed in a GAA deficient cell line. We have demonstrated that the mutation does not prevent normal splicing; low levels of correctly spliced mRNA are generated with the mutant construct. The data explain why the mutation is restricted to a milder, adult-onset phenotype. We also demonstrate that splicing out of exon 2 occurs with the wild-type construct, and thus represents alternative splicing which takes place in normal cells. Three splice variants (SV1, SV2 and SV3) are made with both the mutant and the wild-type constructs. Furthermore, as shown by RNAse protection assay, these mRNA variants are less abundant with the mutant construct. Thus, a major effect of the mutation appears to be a low splicing efficiency, since the total amount of all the transcripts generated from the mutant construct is reduced compared with the wild type. The removal of approximately 90% of the intron 1 (2.6 kb) sequence resulted in a dramatic increase in the levels of correctly spliced mRNA, indicating that the intron may contain a powerful transcriptional repressor.

Adult↗
PubMed1996

Glycogenosis type VII (Tarui disease) in a Swedish family: two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions.

Phosphofructokinase (PFK) plays a major role in glycolysis. Human PFK is composed of three isoenzyme subunits (muscle [Ml, liver [L], and platelet [P]), which are encoded by different genes. Deficiency of muscle isoenzyme (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, Italian, French Canadian, and Swiss patients. We describe the genetic defect in a Swedish family with affected individuals in two generations. The patients are compound heterozygotes: two different mutations result in retention of intron 13 or intron 16 sequences into mRNA. A G1127A transition destroys the 5' donor site of intron 13, resulting in a 155-nt retention of the intronic sequence. An a-to-g base change in intron 16 creates a new acceptor splice site, resulting in a 63-nt retention of intronic sequence. Both mutations are predicted to result in premature termination of translation. Some of the transcripts generated from the intron 16 mutated allele also contain intron 10 sequence unspliced.

Adolescent↗
PubMed1995

Human isoleucyl-tRNA synthetase: sequence of the cDNA, alternative mRNA splicing, and the characteristics of an unusually long C-terminal extension.

The human isoleucyl-tRNA synthetase (IRS)-encoding cDNA, whose primary structure we report here, has an open reading frame (ORF) which encodes a protein of 1262 amino acids (aa) with strong homology to IRS from yeast (53.5%) and Tetrahymena (51.0%) and contains all the major consensus motifs of class-I hydrophobic amino-acyl-tRNA synthetases (aaRS; MRS, LRS, VRS, IRS). However, the human enzyme has an unusually long C-terminal extension composed, in part, of a twice-repeated motif which shows no homology to any reported protein. We also report the presence of a coiled-coil-like motif in the C-terminal half of the protein. The mRNA has an additional exon in the 5'-untranslated region (UTR) which is alternatively spliced, giving rise to two types of mRNA, both of which are expressed in several human tissues. The longer of the two transcripts contains predicted secondary structure in the 5'-UTR which may reduce the translational efficiency of this mRNA. Two possible regulatory elements in the 5'-UTR, an interferon-stimulated response element (ISRE)-like sequence and a short ORF, have been identified. Because human IRS has previously been shown to be the target of antibodies in autoimmune disease, we discuss the role of protein structural features in the development of an autoimmune response to IRS.

Alternative Splicing↗
PubMed1995

Genetic defects in patients with glycogenosis type II (acid maltase deficiency).

Inherited deficiency of acid alpha-glucosidase (acid maltase, GAA) leads to glycogen storage disease type II. Clinical manifestations and prognosis of the disease depend on the age of onset and tissue involvement. GAA deficiency is extremely heterogeneous, ranging from a rapidly progressive fatal infantile-onset form to a slowly progressive adult-onset myopathy associated with respiratory insufficiency. Biochemical and immunochemical studies of the biosynthesis of the enzyme in GAA-deficient patients established the molecular diversity of the disease. Cloning and sequencing of the cDNA and the gene provided the basis for genetic analysis of the patients with different phenotypes. In this article, we summarize the data on mutations in the GAA gene and discuss the correlation between the genotype and phenotypic expression of the disease.

Glucan 1,4-alpha-Glucosidase↗
PubMed1995

Leaky splicing mutation in the acid maltase gene is associated with delayed onset of glycogenosis type II.

An autosomal recessive deficiency of acid alpha-glucosidase (GAA), type II glycogenosis, is genetically and clinically heterogeneous. The discovery of an enzyme-inactivating genomic deletion of exon 18 in three unrelated genetic compound patients--two infants and an adult--provided a rare opportunity to analyze the effect of the second mutation in patients who displayed dramatically different phenotypes. A deletion of Lys-903 in one patient and a substitution of Arg for Leu-299 in another resulted in the fatal infantile form. In the adult, a T-to-G base change at position -13 of intron 1 resulted in alternatively spliced transcripts with deletion of exon 2, the location of the start codon. The low level of active enzyme (12% of normal) generated from the leakage of normally spliced mRNA sustained the patient to adult life.

Adult↗
PubMed1991

Calcium regulation of the human PMN cytosolic 15-lipoxygenase.

Addition of tracer (pg) amounts of [3H]arachidonic acid to the 120,000 x g cytosolic fraction of human polymorphonuclear leukocytes (PMNs) produced [3H]-15-HETE, the product of the 15-lipoxygenase, as the major metabolite. In the presence of nanomolar and low micromolar amounts of calcium, [3H]-15-HETE formation was increased as much as 15-fold which corresponded to 17% conversion of added substrate. This enhancement of the cytosolic 15-lipoxygenase activity, which was reversible by EGTA, was inhibited by phosphatidyl serine and phosphatidyl choline. Millimolar levels of calcium inhibited the cytosolic 15-lipoxygenase and the 5-lipoxygenase product 5-HETE could reverse this inhibition. These results indicate that calcium is an important modulator of the PMN 15-lipoxygenase when the enzyme is in a cytosolic milieu.

Arachidonate 15-Lipoxygenase↗
PubMed1990

5-Hydroxyeicosanoids selectively stimulate the human neutrophil 15-lipoxygenase to use endogenous substrate.

When human neutrophils, prelabeled with [3H]arachidonic acid, were incubated with 5S,15S-dihydroxyeicosatetraenoic acid (5,15-diHETE), a dose-dependent increase in the 15-lipoxygenase product [3H]-15-HETE was observed relative to untreated cells. Typically, a fivefold increase in [3H]-15-HETE formation was obtained upon exposure of these cells to 3 muM 5,15-diHETE. There was no appreciable enhancement of the 5-lipoxygenase metabolite [3H]-5-HETE. Product identities were confirmed by comparing retention times on straight- and reversed-phase HPLC with authentic standards, and RIA. Other 5-hydroxyeicosanoids, such as 5-HETE, 5-HETE methyl ester, and leukotriene B4(5S,12R-diHETE), were equally effective in stimulating the formation of [3H]-15-HETE, but exogenously added lipoxin A4, lipoxin B4, 15-HETE, and 12-HETE were much less potent, whereas stearic acid was ineffective. The diHETEs also showed a greater selectivity in activating the 15-lipoxygenase relative to the 5-lipoxygenase. A likely source of substrate for the 15- and 5-lipoxygenases is a pool of cell-associated but noncovalently bound arachidonic acid. In [3H]arachidonic acid-prelabeled neutrophils, the amount of free [3H]arachidonic acid ranged between 50 and 700 fmol/10(7) cells, whereas unlabeled neutrophils contained 100-2,200 pmol/10(7) cells of nonesterified arachidonic acid. The exogenously added hydroxyeicosanoids induce a 0.5-3% conversion of this substrate pool to product. These findings indicate that the 15-lipoxygenase in human neutrophils is a cryptic enzyme that needs to be stimulated in order to metabolize endogenous substrate. It is possible that 5-hydroxyeicosanoids may mimic an as yet unidentified physiological activator of the 15-lipoxygenase.

Arachidonate 15-Lipoxygenase↗
PubMed1989

The hierarchical structure of geriatric patient satisfaction. An Older Patient Satisfaction Scale designed for HMOs.

This paper describes an instrument design effort aimed at measuring patient-satisfaction among older (65 years and over) subscribers of HMOs. The study was conducted in a multi-satellite prepaid group practice in Buffalo, New York. In order to be able to construct a satisfaction measure that would reflect the interests of the actual consumers of HMO-services, a series of four focused group interviews were held with 24 randomly selected elderly enrollees. The substantive content of these interviews was systematically analyzed for both topics and ideas, yielding a total of 173 distinct ideas about the perceived satisfaction with the services received expressed over 3,176 lines of narrative. From this substantive pool, sixty attitudinal statements were constructed with the ideas represented in these statements being proportional to the number of lines of transcribed discussion devoted to each topic. This 60-item Older Patient Satisfaction Scale (OPSS) was submitted to a systematic sample of 229 elderly HMO subscribers. They also were asked to complete two existing scales: the Ware PSQ, and the Larsen CSQ-8. Factor analysis performed on the OPSS-items yielded 14 primary factors of geriatric patient satisfaction, two second-order and one third order general factor. As the second-order factors accounted for the largest proportion of the common variance, those items of the original 60-item OPSS were identified that had highest loadings on these second-order factors, yielding 7 such items for one and 5 for the other. These scales had alpha-reliabilities of .83 and .80, respectively. It was also found that the OPSS had good convergent validity with the PSQ and CSQ-8. The overall psychometric properties identified for the OPSS, as well as the fact that it was constructed from a health-care consumer's perspective, makes it well suited for use with a unique and rapidly expanding geriatric patient population.

Aged↗