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Biomedical subjects

R E Hall

Publications and source records attributed to R E Hall.

At least 37 records · Page 2Linked to original sources
PubMed1996

cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.

P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-alpha and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3' reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M. hominis, M.iowae, M.synoviae or M.lypophilum. Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primrs revealed Mycoplasma sequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone from M. fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5' end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M. fermentans or a closely related species. P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.

Amino Acid Sequence↗
PubMed1996

Searching for an improved clinical comorbidity index for use with ICD-9-CM administrative data.

We studied approaches to comorbidity risk adjustment by comparing two ICD-9-CM adaptations (Deyo, Dartmouth-Manitoba) of the Charlson comorbidity index applied to Massachusetts coronary artery bypass surgery data. We also developed a new comorbidity index by assigning study-specific weights to the original Charlson comorbidity variables. The 2 ICD-9-CM coding adaptations assigned identical Charlson comorbidity scores to 90% of cases, and specific comorbidities were largely found in the same cases (kappa values of 0.72-1.0 for 15 of 16 comorbidities). Meanwhile, the study-specific comorbidity index identified a 10% subset of patients with 15% mortality, whereas the 5% highest-risk patients according to the Charlson index had only 8% mortality (p = 0.01). A model using the new index to predict mortality had better validated performance than a model based on the original Charlson index (c = 0.74 vs. 0.70). Thus, in our population, the ICD-9-CM adaptation used to create the Charlson score mattered little, but using study-specific weights with the Charlson variables substantially improved the power of these data to predict mortality.

Aged↗
PubMed1996

Expression of the androgen receptor and an androgen-responsive protein, apolipoprotein D, in human breast cancer.

Little is known regarding the activity and function of the androgen receptor (AR) in human breast cancer. In the present study AR was evaluated in untreated primary breast cancers using antisera to the amino- and carboxy-termini of the receptor and quantitated using colour video image analysis. A strong correlation between tissue concentration and percentage AR-positive cells was observed for each antiserum. However, comparison of percentage positive cells using the amino- and carboxy-terminal AR antisera in individual breast cancer specimens revealed a subset of tumours with discordantly increased staining for the carboxy terminus. These findings suggest the presence of amino-terminal-truncated AR in a proportion of breast cancer cells or presence of AR mutations or associated protein alterations that affect binding of the amino-terminal AR antiserum. Immunohistochemical expression of the androgen-regulated glycoprotein, apolipoprotein D (apo-D), was also evaluated in the breast cancer specimens. Focal positivity of apo-D staining, which did not always co-localise with AR-positive cells, was observed within breast tumours. Furthermore, no correlation was evident between percentage positive cells stained for AR and apo-D in breast cancer specimens. These findings indicate that, although apo-D expression is androgen regulated in human breast cancer cell lines in vitro, its expression in primary breast cancers may be regulated by other factors. The expression of AR in primary breast cancers also suggests that the receptor may be involved in tumour responsiveness or in abnormal responses to endocrine therapies.

Adult↗
PubMed1995

Detection and analysis of an alternatively spliced isoform of interleukin-6 mRNA in peripheral blood mononuclear cells.

Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transciptase-polymerase chain reaction (RT-PCR) studies to examine lL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced lL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the lL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed lL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of lL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the lL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of lL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.

Amino Acid Sequence↗
PubMed1995

A comparison of crossover versus parallel-group design in the evaluation of analgesic efficacy after molar extraction.

This study compares the analgesic effects of two standard combinations (Empirin with codeine versus Mersyndol) and placebo as measured by crossover versus parallel-group design. The analgesic results obtained with three groups of 12 to 13 crossed over subjects with two extractions divided into six subgroups of five to seven subjects for each sequence were qualitatively the same and statistically at least as strong as those obtained by analysis of parallel groups of 38 to 42 extractions per group. By both methods the analgesics were statistically significantly more effective than placebo. The difference between the two products was not statistically significant, although the score for Mersyndol was somewhat better by both methods of study. The crossover data did not allow judgments concerning side effects in spite of its greater efficiency in quantifying pain relief.

Acetaminophen↗
PubMed1995

Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3.

Progression to hormone-refractory disease is a common outcome of human prostate cancer. In this study, we have investigated the basis of androgen insensitivity in the human prostate cancer cell line, PC-3, which was derived from a bone metastasis of a hormone-refractory prostate cancer. PC-3 cells with virtually undetectable (PC-3AR-) or low (PC-3AR+) levels of androgen receptor (AR) RNA expression were examined. RNase protection assays demonstrated that the level of AR RNA in PC-3AR+ cells was similar to that in a normal androgen-responsive genital skin fibroblast cell strain. Quantitative immunocytochemical staining of AR in PC-3AR+ cells using antibodies directed against the amino and carboxyl termini of the receptor revealed staining in 30% of cells with either antibody. Furthermore, the level of AR staining in PC-3AR+ cells was higher than in the androgen-responsive breast cancer cell lines ZR-75-1, T47-D, and MCF-7. Despite the expression of AR RNA and protein, PC-3AR+ cell proliferation was unaffected by the presence of 0.1-10 nM mibolerone. Scatchard analysis demonstrated a complete absence of specific [3H]dihydrotestosterone ([3H]DHT) binding to PC-3AR+ cytosolic extracts, which could not be explained by structural alterations in the AR gene. The sizes of individual AR exons amplified from genomic DNA derived from PC-3AR+ cells were identical to those amplified from normal human cells. Furthermore, sequence analysis did not reveal a mutation in the DNA- or hormone-binding domains of the AR gene in PC-3AR+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1995

Up-regulation of cytokine mRNA in human monocytes and myeloid cell lines by the differentiation/activation factor p48.

Polypeptide 48 is a 48,000 MW protein, originally isolated from conditioned media of some human leukaemic cell lines, that induces differentiation and cytolytic activity in HL-60 promyelocytic leukaemia cells and activates human peripheral blood monocytes to secrete interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha). In the present study we examined the effects of p48 on the accumulation of a series of monokine transcripts, including TNF-alpha, IL-1 alpha, IL-1 beta and IL-6, in human peripheral blood monocytes and the myeloid/monocyte cell lines HL-60 and U937. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis, p48 was found to induce accumulation of TNF-alpha, IL-1 alpha and IL-1 beta mRNA in peripheral blood monocytes, HL-60 and U937 cells. IL-6 mRNA was found to be increased in p48-stimulated peripheral blood monocytes but not HL-60 or U937. Thus, the secretion of IL-1 and TNF-alpha by p48-stimulated monocytic cells was associated with up-regulation of cytokine mRNA, suggesting that p48 leads to increased transcription or mRNA stability in these cells. As U937 and HL-60 are likely to represent premonocyte stages of haemopoietic differentiation, it is possible that the effect of p48 on IL-6 mRNA, in contrast to its effect on TNF and IL-1, requires cells to be at a later differentiation step.

Blotting, Northern↗
PubMed1994

Variations in hysterectomy rates in Ontario: does the indication matter?

OBJECTIVES: To examine variations in rates of hysterectomy for the five main indications for the procedure in regions of Ontario. DESIGN: Cross-sectional population-based analysis of hospital discharge abstracts. SETTING: All acute care facilities in Ontario. PARTICIPANTS: All 65,599 women whose hospital record contained a procedure code indicating that a hysterectomy was performed between Apr. 1, 1988, and Mar. 31, 1991. Duplicate cases, records of cancelled procedures and nonresidents were excluded. MAIN OUTCOME MEASURES: Crude and age-adjusted rates of hysterectomy, by indication, for each region of Ontario. RESULTS: Five indications accounted for more than 80% of hysterectomies performed. The median age-adjusted rate of hysterectomy for Ontario regions during the study period was 6.25 per 1000 women, with a 2.7-fold variation among regions. The regions with rates of hysterectomy in the highest quartile tended to be rural, and those with rates in the lowest quartile tended to be urban areas with teaching hospitals. When rates of hysterectomy for specific indications were examined, they showed substantial variations among regions in the rate of the procedure for menstrual hemorrhage (18-fold variation), uterine prolapse (9.3-fold) and endometriosis (6.3-fold). A smaller but still significant variation was shown in the rate of hysterectomy for leiomyoma (2.3-fold). Regional variation in the rate of hysterectomy for cancer (2.5-fold) was not statistically significant. CONCLUSIONS: There are large interregional variations in rates of hysterectomy, especially for indications that are more discretionary than others (i.e., menstrual hemorrhage, uterine prolapse and endometriosis) and less variation in rates when treatment options and diagnosis are clear-cut. This result suggests the need for more definitive practice guidelines on treatment of the indications for which the rate is more variable.

Adult↗
PubMed1994

MDA-MB-453, an androgen-responsive human breast carcinoma cell line with high level androgen receptor expression.

The role of androgens and the androgen receptor (AR) in the development and progression of breast cancer is poorly understood. To further define a potential model for androgen action in breast cancer, MDA-MB-453 cells, which express AR in the absence of oestrogen receptors and progesterone receptors, were further characterised in terms of AR expression and androgen responsiveness. High level expression of AR was confirmed by northern blot analysis, radioligand binding and immunocytochemistry, and could not be accounted for by AR gene amplification. Three endogenous androgen-responsive genes (fatty acid synthetase, gross cystic disease fluid protein of 15 kDa and prolactin receptor) and a transfected reporter gene, containing an androgen-responsive element, were induced following androgen administration. A synthetic androgen, mibolerone, induced moderate (27% above control) stimulation of MDA-MB-453 cell proliferation, which was abrogated by the simultaneous administration of the synthetic androgen antagonist, anandron, demonstrating that the effect was AR-mediated. In summary, MDA-MB-453 cells express high levels of functional AR, and thus provide a valuable in vitro model for further studies on androgen regulation of gene expression, and perhaps cell proliferation in breast cancer.

Blotting, Southern↗
PubMed1993

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies.

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.

Amino Acid Sequence↗
PubMed1993

Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria (1). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51- and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters.

Aconitate Hydratase↗
PubMed1992

Regulation of androgen receptor gene expression by steroids and retinoic acid in human breast-cancer cells.

Although the androgen receptor (AR) has been detected by ligand-binding assays, there is little known about the expression and regulation of the AR gene in human breast-cancer cells. AR mRNA, measured by Northern analysis in 18 cell lines, was found to be expressed predominantly in oestrogen- and progesterone-receptor-positive (ER+, PR+) lines as a single species of approximately 10.5 kb but was also comparatively abundant in I ER- and PR-negative cell line, MDA-MB-453. Dexamethasone (Dex), Organon 2058 (Org 2058), dihydrotestosterone (DHT), and all-trans-retinoic acid (RA) down-regulated AR mRNA levels in T-47D (ER+, PR+) cells 6 hr after treatment, whereas oestradiol (E2) had no effect. In MDA-MB-453 (ER-, PR-) cells, regulation of AR mRNA by RA differed from the other cell lines: RA increased the level of AR mRNA. DHT-binding assays indicated a corresponding increase in AR protein. Transfection of the androgen-responsive mouse mammary tumour virus long-terminal repeat (MMTV LTR) linked to a chloramphenicol acetyltransferase (CAT) reporter gene was used to examine the effect of altered AR levels on androgen action. The increased level of AR following RA pre-treatment in MDA-MB-453 cells resulted in enhanced induction of CAT activity by DHT and, conversely, a decrease in the level of AR following RA pretreatment in T-47D cells resulted in reduced induction of CAT activity by DHT. These data demonstrate that AR is expressed predominantly in ER+ and PR+ cell lines and its expression is regulated by ligands also known to regulate ER or PR, including progestins and retinoids. Androgen responsiveness measured by a transfected reporter gene was altered according to the extent of up- or down-regulation of AR expression, demonstrating that responsiveness is dependent on receptor concentration.

Breast Neoplasms↗
PubMed1992

Expression and regulation of retinoic acid receptors in human breast cancer cells.

Retinoic acid is known to inhibit mammary carcinogenesis in rodents and to inhibit proliferation and steroid hormone receptor gene expression in human breast cancer cells. Since these effects are likely to be mediated by nuclear retinoic acid receptors (RARs) the present study was initiated to determine the expression and regulation of RARs in human breast cancer cell lines. Differential cellular gene expression of the RARs was determined by Northern blot analysis of total RNA prepared from 5 ER+ and 6 ER- cell lines. RAR alpha was detected as mRNA species of 2.7 and 3.4 kilobases in all cell lines and the level of gene expression was greater in ER+ cell lines (P less than 0.001). RAR beta mRNA (3.7 kilobases) was detected in seven of the eleven lines tested and was expressed most commonly in ER- cell lines. RAR gamma mRNA was expressed in all cell lines as a transcript of 3.4 kilobases at levels that were similar in both ER+ and ER- cell lines. Retinoic acid failed to regulate the expression of the RAR alpha and RAR gamma genes. The effect of steroid hormones on RAR alpha and RAR gamma mRNA levels was also examined. In four PR+ cell lines (T-47D, BT 474, MCF-7M, and MDA-MB-361), progestins markedly decreased RAR alpha mRNA levels. The progestin effect on RAR alpha levels in T-47D cells was detectable at concentrations of 0.05 nM and was maximal at 1 nM 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione ORG 2058, whereas dihydrotestosterone and dexamethasone were without effect. RAR alpha and RAR gamma mRNA levels were rapidly decreased by progestin, and the effect was maximal 3-6 h after ORG 2058 treatment. However, the mRNA loss was transient, and recovery of RAR alpha and RAR gamma mRNA levels was noted 12-24 h after retinoic acid treatment. Although RAR gamma mRNA returned to control levels by 24 h, the decrease in RAR alpha mRNA was maintained at around 50% control until at least 48 h. In summary, RAR alpha and RAR gamma were expressed in all human breast cancer cell lines and were regulated by progestins in the PR+ T-47D cell line. The previously reported ability of retinoic acid to down-regulate PR mRNA and the present demonstration that progestins down-regulate RAR alpha and RAR gamma mRNA suggest that mutual regulation may be a mechanism through which PR and the RARs interact in human breast cancer cells.

Breast Neoplasms↗
PubMed1992

Expression of the adhesion molecules ICAM-1 and ICAM-2 on tumor cell lines does not correlate with their susceptibility to natural killer cell-mediated cytolysis: evidence for additional ligands for effector cell beta integrins.

The LFA-1 molecule (CD11a/CD18), a member of the leukocyte (beta 2) integrin subfamily of the integrin supergene family, has been shown to subserve important function(s) in natural killer (NK) and lymphokine-activated killer (LAK) effector cells based on monoclonal antibody inhibition and other studies. Presently, two cellular ligands for LFA-1 have been identified, termed ICAM-1 and ICAM-2. In this study, we have examined the role of target cell ICAM-1 (CD54) and ICAM-2 in NK-mediated target lysis. Using a panel of tumor target cell lines, ICAM-1 surface protein and transcript expression did not correlate with sensitivity to NK lysis. Compared to ICAM-1, ICAM-2 transcript expression was very low or undetectable in tumor cell targets, and also did not correlate with sensitivity to NK lysis. ICAM-1+ K562 cells and K562 cells which were rendered surface ICAM-1- with an antisense oligonucleotide were equally sensitive to NK lysis. Finally, human ICAM-1- P815 cells were stably transfected with the human ICAM-1 gene, and both ICAM-1- P815 (wild type) and ICAM-1+ stable transfectants were equally insensitive to NK lysis. These studies provide evidence that ICAM-1 and ICAM-2 are not important target cell ligands for NK effector cell LFA-1 and that other target cell ICAM may exist.

Animals↗
PubMed1992

Human p150,95 alpha-subunit: genomic organization and analysis of the 5'-flanking region.

LFA-1, Mac-1, and p150,95 comprise a family of cell-surface glycoproteins that mediate adhesive interactions of myeloid and lymphoid cells. These glycoproteins are heterodimers composed of a common beta-subunit and distinct alpha-subunits. The chromosomal gene for the alpha-subunit of p150,95 was isolated to provide a framework from which to study the mechanisms for expression of the gene. The gene spans 30 kb of DNA and contains 31 exons. In agreement with a previous report by Corbi et al. (1990), the exons were found to be divided into five groups separated by large introns. The extracellular domains are encoded in exons 2 through 30 while the transmembrane and cytoplasmic domains are encoded in exons 30 and 31. We have expanded these findings in a number of ways. The first exon contains the 5' untranslated region. The 2,163-bp 5'-flanking sequence contains the first intron and several putative transcriptional initiation sites preceded by two TATA sequences and two GC-like boxes. Additional sequence motifs for a variety of DNA-binding proteins are present and discussed. Fusions of the bacterial chloramphenicol acetyltransferase gene (CAT) to approximately 5.3 kb of 5'-flanking DNA and also to subcloned fragments of this region were constructed and transfected into the human promonocytic cell line, U937. CAT expression was inducible with phorbol-12-myristate-13-acetate (PMA) and full expression was dependent on the presence of intron 1 and sequences upstream from the 2,163-bp flanking DNA. Additionally, intron 1 and a region further upstream contain functional cis-acting sequences.

Base Sequence↗
PubMed1991

Differentiation-inducing cytokine P48 exists in a membrane-associated form.

P48 is a recently described 48-kDa differentiation-inducing cytokine isolated from the culture medium of the human leukemia line Reh. P48 induces differentiation and cytolytic activity in the promyelocytic cell line HL-60, and stimulates the release of TNF-alpha and IL-1 from peripheral blood monocytes. In further studies designed to examine the biosynthesis and function of P48, surface immunofluorescence flow cytometry analysis as well as 125I surface labeling and immunoprecipitation, revealed the presence of P48 on the surface of Reh cells. Triton X-114-treated Reh cells were partitioned into detergent and aqueous phases and separated by SDS-PAGE. Western blot analysis revealed that P48 partitioned exclusively into the detergent phase, suggesting an integral membrane association. Reh cells fixed with paraformaldehyde, but not K562 or P815, were able to stimulate the release of TNF-alpha from peripheral blood monocytes in a manner similar to that of secreted P48. Isolated plasma membranes from Reh cells could also stimulate TNF-alpha release. This TNF-alpha-releasing activity could be removed from detergent solubilized Reh membranes by immunoaffinity chromatography on an anti-P48 column. This study suggests that, in addition to being secreted into the culture medium, P48 is expressed on the surface of Reh cells in a biologically active form. The membrane form of P48 may be 1) a final maturation step before secretion or 2) a cell membrane-associated form that may be analogous to the membrane forms of TNF-alpha and IL-1.

Blotting, Western↗