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Biomedical subjects

G E Austin

Publications and source records attributed to G E Austin.

At least 19 recordsLinked to original sources
PubMed2002

Functional activity of three distinct myeloperoxidase (MPO) promoters in human myeloid cells.

The enzyme myeloperoxidase (MPO) is synthesized only in myeloid and monocytic cells, making it an important marker of the myeloid lineage. Transcription of the MPO gene is turned on early during the myeloblast stage of myeloid differentiation and is turned off when myeloid precursors are induced to differentiate along any one of a number of pathways. MPO transcripts show heterogeneity in size and sequence due, in part, to differential RNA splicing. We recently reported transfection studies which showed the presence of three distinct MPO promoters in the 5'-flanking region of the human MPO gene, suggesting that MPO transcription may also be regulated through the use of multiple promoters. We now report results of primer extension and RT-PCR experiments designed to determine if transcription of the human MPO gene is initiated at multiple promoter sites in vivo. MPO RNA obtained from myeloid cell lines was analyzed by primer extension using primers located at various sites between bp -1100 and bp +120 of the MPO gene. In addition, RT-PCR experiments were carried out using primer pairs located at intervals between bp -1000 and bp +2500 of the MPO gene. MPO transcripts were found to be initiated at three sites located about bp -920, bp -310, and bp +1 of the MPO gene, corresponding closely to our previously described P3, P2 and P1 promoters, respectively. Transcription initiated at the P1 site gave rise to large transcripts and showed the expected downregulation following induction of differentiation. On the other hand, transcripts initiated at the P3 and P2 sites did not show downregulation following induction of macrophage differentiation by TPA, and most did not appear to extend into the MPO coding region. Northern blot analysis of transcripts initiated at the P3 and P2 sites suggested that transcription at these sites was non-tissue-specific and indicated that many of these transcripts undergo premature termination. These results demonstrate that the MPO gene is transcribed in vivo primarily using the P1 promoter and that the low level of transcription occurring at the P2 and P3 sites is nonspecific and does not contribute significantly to physiologic regulation of MPO gene expression.

5' Flanking Region↗
PubMed2001

Cloning and expression of human HBP1, a high mobility group protein that enhances myeloperoxidase (MPO) promoter activity.

Factors which regulate transcription in immature myeloid cells are of great current interest for the light they may shed upon myeloid differentiation. In the course of screening for transcription factors which interact with the human myeloperoxidase (MPO) promoter we, for the first time, identified and cloned the cDNA and genomic DNA for human HBP1 (HMG-Box containing protein 1), a member of the high mobility group of non-histone chromosomal proteins. HBP1 cDNA was initially cloned from rat brain in 1994, but its presence in human cells or in myeloid tissue had not been described previously. The sequence of human HBP1 cDNA shows 84% overall homology with the rat HBP1 cDNA sequence. We have subsequently cloned the gene, which is present as a single copy, 25 kbp in length. Northern blotting reveals a single 2.6 kb mRNA transcript which is expressed at higher levels in human myeloid and B lymphoid cell lines than in T cell lines tested and is present in several non-myeloid human cell lines. Comparison of the mRNA and genomic sequences reveals the gene to contain 10 exons and 9 introns. The sequence of human HBP1 mRNA contains a single open reading frame, which codes for a protein 514 amino acids in length. The amino acid sequence specified by the coding region shows 95% homology with the rat HBP1 protein. The human protein sequence exhibits a putative DNA-binding domain similar to that seen in rat HBP1 and shows homology with the activation and repressor domains previously demonstrated in the rat protein. We have expressed human HBP1 protein both in vitro and in prokaryotic and eukaryotic cells. The expressed fusion protein binds to a sequence in a functionally important region within the basal human MPO promoter. In transient co-transfection experiments HBP1 enhances MPO promoter activity. Human HBP1 appears to be a novel transcription factor which is likely to play an important role in regulating transcription in developing myeloid cells.

Amino Acid Sequence↗
PubMed2000

Myeloid metaplasia presenting as a splenic mass: report of a case with fine-needle aspiration cytology.

Myeloid metaplasia, i.e., the formation of bone marrow elements outside of the medullary cavity, may occur in any patient with anemia, regardless of the etiology of the anemia itself. The spleen is frequently the site of myeloid metaplasia, and it may present with varying degrees of splenomegaly; however, no cases have been reported where this entity presented as an isolated splenic mass. Our case was a 79-yr-old man who presented with anemia and a splenic mass by CT scan and who underwent fine-needle aspiration (FNA) of that mass. The smears revealed megakaryocytes admixed with other hematopoietic elements. No fatty elements were identified in the smears. We conclude that splenic myeloid metaplasia may present as a discrete splenic mass lesion, and may be diagnosed using the FNA technique.

Aged↗
PubMed2000

Prevalence of hepatitis C virus seropositivity among hospitalized US veterans.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in the United States and abroad. HCV antibody prevalences ranging from 10 to 90% have been reported in intravenous drug abusers, hemodialysis patients, and persons suffering from other liver diseases, whereas HCV seropositivity rates for volunteer-blood donor populations are generally under 1%. However no information has been available concerning the prevalence of HCV in general hospital populations in the United States. METHODS: We examined the rate of HCV seropositivity in 530 patients admitted to the Atlanta VA Medical Center between November 1993 and November 1994. The test population consisted of 400 random hospital admissions, 100 successive admissions to the surgical service, and 30 random admissions to the gastrointestinal service. Serum samples were assayed for HCV antibodies by a second generation EIA, and all repeat reactives were re-examined using a supplemental research assay to confirm the presence of HCV antibodies. Complete chart reviews were carried out on all HCV seropositive patients and on 100 HCV seronegative patients. RESULTS: Sixty-two of the 530 patients tested (11.7%) were repeatedly positive for HCV antibodies. Of these 62 repeat reactives, 56 (90.3%) were positive and 3 others (4.8%) indeterminate by the supplemental assay. The HCV seropositivity rate after supplemental testing was 11.8% for random admissions, 5.0% for surgical admissions, and 13.3% for patients admitted to the gastroenterology service. HCV-associated risk factors in HCV seropositive patients included a history of intravenous drug abuse, current or previous alcohol abuse, previous or concurrent liver disease, previous blood transfusions, hemodialysis, and multiple sex partners or unsafe sex. CONCLUSIONS: HCV infection may be more prevalent among hospitalized VA patients (and among other US hospital populations) than previously expected.

Adult↗
PubMed2000

Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites.

Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the "gold standard" for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS. Blastocystis hominis and Iodamoeba bütschlii were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E. histolytica, E. coli, and C. mesnili. Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA.

Animals↗
PubMed1999

Usefulness of fructosamine for monitoring outpatients with diabetes.

BACKGROUND: Measurements of total glycohemoglobin (glycoHb) or hemoglobin A1c are routinely used to evaluate intermediate-to-long term glycemic control in patients with diabetes. However, despite the recent availability of more rapid methods for glycohemoglobin determination, it remains difficult in many institutions to obtain same-day glycoHb determinations in time to assist physicians with management of outpatients with diabetes. Hence, we investigated whether fructosamine, which reflects very recent (2 to 3 weeks) glycemic control and which can be assayed more rapidly in our laboratory, could serve as a useful adjunct to glycoHb for management of these patients. METHODS: Diabetes control in outpatients managed using fructosamine, fasting serum glucose, and glycoHb (concentrations from the prior visit) was compared with that for outpatients monitored using fasting serum glucose and prior glycoHb alone. The relative usefulness of fructosamine, current and prior glycoHb, and "fasting" serum glucose for evaluation and management of outpatients with diabetes was compared. In addition, the acceptance of fructosamine by physicians was evaluated by a questionnaire. RESULTS: Same-day fructosamine concentrations correlated better with current glycoHb than did either prior glycoHb or current fasting serum glucose concentrations. However, the availability of same-day fructosamine results did not objectively improve diabetes control compared with that obtained using only fasting serum glucose values and prior glycoHb concentrations. Nonetheless, most examining physicians stated that same-day fructosamine concentrations helped them significantly with diabetes management, primarily because many patients do not adequately monitor home blood glucose levels. CONCLUSIONS: Fructosamine may be a useful adjunctive test for management of outpatients with diabetes in situations where it is not practical to obtain same-day glycoHb concentrations, for patients less compliant with home glucose monitoring, or where recent changes in insulin dose or clinical presentation might not be reflected in the glycoHb levels.

Blood Glucose↗
PubMed1998

Identification of an upstream enhancer containing an AML1 site in the human myeloperoxidase (MPO) gene.

Myeloperoxidase (MPO) is an important antibacterial enzyme found only in granulocytes and monocytes. The human MPO gene is transcribed early during myelogenesis but MPO RNA synthesis ceases at the end of the promyelocyte stage of myeloid maturation. We recently identified a basal MPO promoter and several adjacent cis-elements in the proximal 5'-flanking region of this gene. Transfection studies using constructs containing several kb of 5'-flanking MPO DNA revealed the presence of a DNA segment located between bp (base pair) -4200 and bp -3800 with enhancer activity for the endogenous basal MPO promoter. Deletion studies revealed the core enhancer activity to lie between bp -4100 and bp -3844. The percentage enhancement of promoter activity is greater in MPO-expressing myeloid cells than in MPO-non-expressing myeloid cells or non-myeloid cells. Furthermore. the enhancer confers TPA- or DMSO-responsiveness upon either endogenous or exogenous promoters. DNase I footprinting and transfection experiments identified an AML1 site as a functionally important element within the enhancer. Gelshift competition and supershift experiments demonstrated the binding of the alpha subunit of the transcription factor AML1 to this site in HL-60 cells. This distal enhancer appears likely to play an important role in the control of MPO transcription during myeloid differentiation.

Base Sequence↗
PubMed1998

Prevalence of myeloperoxidase gene expression in infant acute lymphocytic leukemia.

The enzyme myeloperoxidase (MPO; donor: H2O2 oxidoreductase, EC1.11.1.7) is a well-established marker of myeloid differentiation. Most myeloid leukemias express MPO enzyme activity at the light microscopic level, whereas lymphoid leukemias characteristically lack such expression. However, the diagnostic significance of MPO RNA expression or of immunohistochemically detectable MPO protein expression in leukemic blasts is unclear. We studied the prevalence and diagnostic significance of MPO RNA and protein expression in 57 cases of MPO enzyme-negative infant B-precursor acute lymphocytic leukemia (ALL), since the blast cells in this condition have been reported to show a high incidence of coexpression of myeloid-associated antigens. MPO expression was compared with other clinical and laboratory parameters. Of the cases examined, 56% showed detectable MPO expression at the RNA or protein level or both. Most positive cases showed MPO protein in many leukemic blasts, whereas a few cases showed substantial MPO protein expression in only a few blast cells. MPO expression showed no significant correlation with other markers of myeloid differentiation. Leukemic lymphoblasts in infant ALL frequently express MPO at the RNA or protein level; this expression does not imply an overall myeloid phenotype. The leukemic blasts in infant ALL may derive from an immature hematopoietic precursor cell not fully committed to lymphoid differentiation.

Blotting, Northern↗
PubMed1998

Myeloperoxidase gene expression in infant leukemia: a Pediatric Oncology Group Study.

A high incidence of co-expression of myeloid-associated antigens in infant B-precursor Acute Lymphocytic Leukemia (B-ALL) has been reported, but the significance of this finding is uncertain. To further assess myeloid differentiation and its prognostic significance in this disease, we investigated the frequency of myeloperoxidase (MPO) gene expression in the blast cells from 43 infants with B-ALL registered in a Pediatric Oncology Group (POG) Pilot Study for Treatment of Infant ALL, utilizing a molecular probe for detection of MPO messenger RNA (mRNA) by Northern blot hybridization and a monoclonal antibody to detect MPO-protein by immunohistochemical staining. Sufficient RNA for Northern blot was extracted from 32 bone marrow or blood samples. In two cases, MPO mRNA was determined by a reverse transcriptase-polymerase chain reaction assay and was negative in both cases. MPO-specific transcripts (MPO+) were present in 19 of 34 (56%) samples analyzed. Immunoreactive MPO protein was positive in 13 of the 20 (65%) patients studied. No correlation was found between MPO gene expression and clinical or laboratory features, karyotypic patterns or clinical outcome. The high frequency of MPO gene expression demonstrated in this study suggests that leukemogenic events in many cases of infant B-ALL appear to involve a pluripotent stem cell not yet fully committed to lymphoid differentiation.

Antibodies, Monoclonal↗
PubMed1997

Identification and functional analysis of multiple murine myeloperoxidase (MPO) promoters and comparison with the human MPO promoter region.

Myeloperoxidase (MPO) is an important component of the oxidative antibacterial defense system of granulocytes. Mammalian MPO gene expression has been most extensively studied in human and murine cells. Transcription of the human MPO gene appears to begin at a single initiation site and we have recently described the isolation and characterization of the corresponding human MPO promoter. On the other hand, MPO transcripts in murine myeloid cells show several distinct 5'-termini, suggesting the existence of multiple murine MPO promoters. However, significant levels of endogenous murine MPO promoter activity have not been demonstrated heretofore, although several murine MPO enhancers have been described. We now report the identification and preliminary functional characterization of four distinct murine MPO promoters. Sequence comparison of the human and murine MPO promoter regions reveals homologues of three out of four of these murine promoters within the human MPO gene. However, only one of these sites appears to be functionally active in human myeloid cells, possibly because of the interposition of Alu sequences between the putative promoter sites in the human gene.

Animals↗
PubMed1996

Intravenous vs. intraprostatic administration of N-methyl-N-nitrosourea to induce prostate cancer in rats.

To develop an improved model of human prostate cancer, 16-wk-old Wistar rats were treated orally for 18 days with the antiandrogen, flutamide (50 mg/kg body weight [BW]/day), followed by 3 days of s.c. testosterone (100 mg/kg BW). There were the only treatments the control animals received (Group 1, n = 10). On the day after the third testosterone injection, N-methyl-N-nitorsourea (MNU) was administered via the tail vein at a dose of 50 mg/kg BW (Groups 2, n = 40 and 3, n = 20); in some rats, a second dose was delivered by the same route 22 wk later (Group 3). A smaller dose of MNU (15 mg/kg BW) was administered intraprostatically (Group 4, n = 20) to a fourth group. In Groups 2, 3, and 4, silastic capsules containing testosterone were implanted s.c. approximately every 6 wk beginning 1 wk post-MNU. Accessory sex gland tumors arose in MNU-treated rats in Group 2 (12/40, 30%). Group 3 (8/20, 40%), and Group 4 (8/20, 40%); 90% were macroscopic (25/28). There were no neoplasms in these organs in the control rats (Group 1, 0/10). These accessory sex gland neoplasms were adenocarcinomas or undifferentiated carcinomas which appeared to be derived from the prostate based on location and histological characteristics, although the size and spread of some of the tumors precluded definitive localization of the tissue of origin. The incidence of these neoplasms was similar in rats given a single dose of MNU intraprostatically or two doses of MNU i.v., but the animals treated intraprostatically maintained higher body weights and developed fewer extraneous tumors. The average (+/- SD) latent period for clinical or postmortem detection of prostate neoplasia after MNU was shortest in the rats given two i.v. doses (39 +/- 3 wk) compared with the single i.v. dose (45 +/- 6 wk) or an intraprostatic dose (56 +/- 7 wk). In 57% of the cases (16/28), the prostate tumors metastasized to distant sites. An activating point mutation was detected in codon 12 of the Ki-ras oncogene in the MNU-induced primary prostate tumors (8/10 examined), and metastases arising from these prostate tumors (2/3) but was absent in normal prostate tissue (0/6). This study demonstrates that two systemic doses of MNU increase the incidence and decrease the latency of prostate neoplasms compared with a single dose, and that a single dose of MNU injected intraprostatically induces prostate adenocarcinoma without many of the other tumors and weight loss typically found after i.v. administration.

Adenocarcinoma↗
PubMed1996

Cis-elements in the promoter region of the human myeloperoxidase (MPO) gene.

We recently reported the identification and initial characterization of the human myeloperoxidase (MPO) promoter. The minimal or basic MPO promoter lies within the proximal 128 bp of the 5'-flanking region of the MPO gene. Plasmids containing progressively larger segments of the 5'-flanking region show correspondingly greater MPO promoter activity and increased tissue specificity compared with smaller promoter fragments. These findings suggested the presence of a multiple important regulatory cis-elements in the 5'-flanking region of the MPO gene. We now report results of studies which reveal the presence of seven discrete nuclear protein binding sites (DP1-DP7) within the proximal 600 bp of 5'-flanking MPO DNA. DNase I footprinting and gel shift analyses indicate tissue-specific and/or maturation-specific differences in nuclear protein binding to most of these sites, suggesting that they play a role in transcriptional regulation. Mutation of site DP7 stimulates the activity of a 594-bp MPO promoter construct in transfection studies, whereas mutation of any of the six other sites (DP1-DP6) reduces promoter activity. These results indicate that oligonucleotides DP1-DP7 constitute cis-elements which contribute to the activity of the human MPO promoter.

Base Sequence↗
PubMed1996

Short gut syndrome: treatment by neovascularization of the small intestine.

Surgical treatment for short bowel syndrome has been directed toward slowing intestinal transit or increasing the absorptive surface area of the bowel. In the present work, we attempted to enhance bowel absorption by increasing vascularity, using the omentum's unique ability to revascularize incorporated tissue. After a 90% resection of small bowel with primary anastomosis in 5 mongrel dogs, an omental flap based on the right gastroepiploic vessels was incorporated into a seromuscular incision on the antimesenteric border of the remnant small bowel. Five control dogs underwent a similar resection and seromuscular incision with an omentectomy. Serum d-xylose assays, hemoglobin, and total protein levels were measured preoperatively and at 3, 6, 9, and 12 weeks postoperatively. Weekly weights were followed. The mean percent d-xylose absorption in the experimental group increased from 96% of the baseline at 3 weeks to 136%, 163%, and 179% at 6, 9, and 12 weeks respectively (p < 0.5). The control group maintained absorption levels between 54% and 74%. Weight loss in the experimental group was significantly less than the controls at weeks 6 and 9, but by 12 weeks, weights were similar for the two groups. Small bowel absorption of d-xylose was significantly enhanced by incorporation of the omentum into the bowel remnant.

Animals↗
PubMed1995

Identification and characterization of the human myeloperoxidase promoter.

Myeloperoxidase (MPO) is a microbicidal protein present in the primary granules of myeloid cells. Transcription of the MPO gene is turned on only during the late myeloblast and promyelocyte stages of myeloid maturation. Identification of cis-regulatory elements and transcription factors which regulate the MPO gene should, therefore, shed light on myeloid maturation. We report transfection and in vitro transcription experiments which demonstrate promoter activity in the proximal 5'-flanking region of the human MPO gene. Using a chloramphenicol acetyl transferase (CAT) reporter vector system, and segments of the 5'-flanking MPO DNA, we constructed a series of MPO promoter-CAT expression vectors. By electroporation and lipofectin-mediated transient transfection assays, as well as by in vitro transcription studies, a 594-bp MPO DNA sequence (bp -583 to +11) showed promoter activity in a variety of MPO-expressing and non-MPO-expressing cell lines. Compared with the SV40 early promoter, the MPO promoter had greater relative activity in MPO-expressing than in non-MPO-expressing cell lines, suggesting slight tissue specificity. However, a CAT reporter plasmid containing 1099-bp of 5'-flanking MPO DNA showed greater specificity for MPO expressing cell lines. Analysis of a group of promoter deletion mutants showed that the minimal promoter was contained in a DNA fragment extending from bp-128 to +11. The remainder of the promoter region contained several segments which appeared to enhance the activity of the minimal promoter. One such enhancer sequence was homologous to an enhancer previously described in the human elastase promoter. Activity of the 594-bp MPO promoter in HL-60 was reduced by only approximately 30% following treatment of the cells with chemical inducers of maturation, but the 1099-bp MPO promoter showed 60% reduction in activity after DMSO treatment. A previously described enhancer region in intron 9 of the MPO gene had little or no effect on activity of the 594-bp MPO promoter. The availability of the MPO promoter will facilitate determination of other factors involved in the regulation of this myeloid-specific gene.

Base Sequence↗
PubMed1994

Mechanical properties of vacuum-mixed acrylic bone cement.

The thrust of the present work was the experimental determination of the uniaxial static compressive and fully reversed tension-compression fatigue properties of CMW 3 acrylic bone cement whose constituents were mixed in a proprietary chamber while simultaneously subjected to a vacuum. Selected indices of performance in this material are: mean static compressive strength, 81.4 MPa; mean compressive modulus of elasticity, 1.95 GPa; endurance limit, 8.1 MPa; and characteristic fatigue life (using a three-parameter Weibull fit to the fatigue test data obtained at a stress of +/- 10 MPa), 238,712 cycles. The difficulties in comparing results obtained using different cement formulations, preparation conditions, and test conditions are detailed. With this in mind, it is suggested that the present results are within the range of values reported by previous workers for other formulations mixed using a variety of methods. The clinical significance of the present results is discussed.

Acrylates↗
PubMed1994

Myeloperoxidase gene expression in normal granulopoiesis and acute leukemias.

Myeloperoxidase (MPO) is an abundant heme protein found in granulocytes and monocytes, which plays an important role in host defense against infection. MPO enzyme activity as determined by light microscopic cytochemistry has long been an important marker used in the diagnosis of acute leukemias and other hematopoietic disorders. Recently, MPO expression has been studied at the electron microscopic level, and monoclonal antibodies (mAbs) against MPO protein have been developed. Furthermore, techniques and probes for analysing MPO expression at the RNA level are now available. This has made possible more extensive studies of MPO expression in a wide range of neoplastic and preneoplastic blood disorders. This review will discuss the fundamental biology of MPO as well as recent developments in our understanding of MPO expression in leukemic cells and cell lines of various lineages.

Acute Disease↗