Biomedical subjects

A C Johnson

Publications and source records attributed to A C Johnson.

At least 37 records · Page 2Linked to original sources

PubMed2001

An upstream repressor element plays a role in Igf2 imprinting.

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.

Animals↗

PubMed2001

Current status of the AbioCor implantable replacement heart.

BACKGROUND: The AbioCor implantable replacement heart (IRH) has been developed as an alternative to transplant (ie, destination therapy). We report our experience with the AbioCor IRH in a bovine model at the University of Louisville. METHODS: Male Holstein cows were used (85 to 115 kg). The internal controller, battery, and secondary transcutaneous energy transfer coil were implanted in the right flank. After cardiopulmonary bypass, the thoracic unit was implanted orthotopically. After removal of air and weaning from cardiopulmonary bypass, the AbioCor was connected to internal components and energy transfer through transcutaneous energy transfer coils was achieved. RESULTS: Nineteen animals underwent implantation of the AbioCor IRH for a proposed 30-day duration. There were 6 deaths, none related to device malfunction. All animals demonstrated normal hemodynamics with normal pressures in the aorta, pulmonary artery, left atrium, and right atrium. There was no significant hemolysis and all animals demonstrated normal end organ function. The internal battery allowed for brief periods of untethered mobility. CONCLUSIONS: The AbioCor IRH has resulted in normal hemodynamics and normal end organ function without evidence of hemolysis in a bovine model.

Animals↗

PubMed2001

Limitations on the role of incorporated organic matter in reducing pesticide leaching.

The use of organic amendments has been suggested as a method of controlling pesticide leaching through soils. The enarenados soils of the intensive horticulture of the Almeria province of southern Spain contain buried organic matter horizons above a soil layer amended with clay. This region is ideal for understanding the potential for and limitations of organic amendments in preventing pesticide pollution. This study measured the sorption and degradation potential of carbofuran in this soil system and the hydrological behaviour of the soil horizons. The sorption of carbofuran was controlled by the organic carbon content, the degradation was strongly pH-dependent and the acidic organic layer protected the sorbed carbofuran against degradation. Hydrologically, the soil system is dominated by ponding above an amended clay layer and by the presence of macropores that can transport water through this clay. A simple model is proposed on this basis and shows that although high levels of dissolved organic carbon can be released by buried organic horizons, the major control on re-release of sorbed pesticide is the potential for sorption hysteresis in this organic layer. A comparison of sorption and degradation data for carbamate insecticides used in the region with groundwater observations for these compounds shows that no amount of incorporated organic would protect against pollution from highly water-soluble compounds.

Biodegradation, Environmental↗

PubMed2001

Screening for WBC antibodies by lymphocyte indirect immunofluorescence flow cytometry: superior to cytotoxicity and ELISA?

BACKGROUND: Cytotoxic WBC antibodies are found in patients who have refractoriness to platelet transfusion (RPT) or are experiencing febrile transfusion reactions (FTRs) and in sera giving so called nonspecific hemagglutination by IAT (N/S IAT). Sera from such patients were screened for WBC antibodies regardless of the ability to fix complement using a flow cytometric (FC) lymphocyte indirect immunofluorescence test (LIFT) to compare FC-LIFT with a routine lymphocytotoxicity test (LCT) for WBC antibody detection. STUDY DESIGN AND METHODS: Serum from 104 patients with RPT, 87 with FTR, and 147 with N/S IAT were tested in parallel by using FC-LIFT and LCT. Sera giving discrepant results were re-tested with an HLA class I antibody ELISA to assess whether they were HLA-specific. RESULTS: Of the sera tested, 175 were LIFT positive, and 146 were LCT positive. Fifty-five had antibodies that were detectable only by LIFT; 26 were positive only by LCT. Of these 81 discrepant sera, 30 of 63 were positive in HLA ELISA. CONCLUSION: FC-LIFT detects more WBC antibodies than does LCT or ELISA, and it is a superior screening technique. Because some cytotoxic antibodies are detectable only by LCT, comprehensive WBC antibody screening would require the application of both techniques. However, because FC assessments of cytotoxicity have been described, LCTs may become redundant for WBC antibody screening.

Antibodies↗

PubMed2001

Avalanche trauma and closed head injury: adding insult to injury.

OBJECTIVE: The incidence of fatal closed head injury (CHI) and nonfatal CHI causing an altered level of consciousness in avalanche victims is unknown. The purpose of this study was to assess the incidence and potential significance of CHI in avalanche-related deaths. METHODS: The records of the state medical examiner and hospital records of all victims killed in avalanches in the state of Utah from October 1, 1992 through April 30, 1999 were reviewed for a cause of death and for the presence of CHI. Closed head injury was described as "present" or "severe," depending on whether the degree of CHI was sufficient to have caused or directly contributed to death, as determined by the medical examiner. RESULTS: In this review, 28 avalanche-related deaths were identified, of which 22 (79%) were due to asphyxia. Seventeen victims (61%) had evidence of some degree of CHI. Six victims (21%) had evidence of severe CHI. One of 7 snowmobile riders sustained a severe CHI, whereas 4 of 16 skiers or snowboarders sustained a severe CHI (P = not significant). CONCLUSION: Although asphyxiation was the cause of death in most avalanche victims, evidence of CHI was present in 61% of the cases studied. While avalanche-associated CHI may not be sufficient to cause death in many cases, a depressed level of consciousness might render a victim incapable of self-rescue and predispose to asphyxia. Helmet use may help prevent avalanche-associated CHI and thus be a useful safety adjunct.

Asphyxia↗

PubMed2000

The Wilms' tumor gene product WT1 mediates the down-regulation of the rat epidermal growth factor receptor by nerve growth factor in PC12 cells.

Recently, we characterized the rat epidermal growth factor receptor (EGFR) promoter and demonstrated that TCC repeat sequences are required for the down-regulation of EGFR by nerve growth factor (NGF) in PC12 cells. In this study, we report that the Wilms' tumor gene product WT1, a zinc finger transcription factor, is able to enhance the activity of the rat EGFR promoter in cotransfection assays. Gel mobility shift assays demonstrate that WT1 binds to the TCC repeat sequences of the rat EGFR promoter. Overexpression of WT1 resulted in up-regulation of the expression levels of endogenous EGFR in PC12 cells. Interestingly, NGF down-regulated the expression levels of WT1 and EGFR in PC12 cells, but not in the p140(trk)-deficient variant PC12nnr5 cells or in cells expressing either dominant-negative Ras or dominant-negative Src. Most importantly, we evaluated the inhibitory effect of antisense WT1 RNA on EGFR expression, and we found that antisense WT1 RNA could substantially reduce EGFR repression in either histochemical staining study or immunoblot analysis. These results indicate that NGF-induced down-regulation of the EGFR in PC12 cells is mediated through WT1 and that WT1 may play an important role in the differentiation of nerve cells.

Animals↗

PubMed2000

Estimating steroid oestrogen inputs into activated sludge treatment works and observations on their removal from the effluent.

A method to predict steroid oestrogen inputs into sewage works is described and tested against available data. For oestradiol (E2), 68% of the predictions were within 50% of the actual measured value, and for oestrone (E1), 52% of the estimations were within 50% of the measured value. Predictions for ethinyloestradiol (EE2), which are particularly sensitive to assumptions on the number of people taking the oral contraceptive, were less accurate. Five Italian and three Dutch activated sludge treatment works (STW) were sampled on two to three occasions for E2, E1, EE2 and E3 (E3 only in Italy) in both influent and effluent waters. High concentrations of E3 were found in the influent, as predicted, with a mean of 57 ng/1 and a mean of 10 ng/l in the effluent. Using the currently available data collected from composite samples, an average of 88% of E2 and 74% of E1 would appear to be removed by the activated sludge process.

Environmental Monitoring↗

PubMed2000

Cloning and characterization of the promoter region of the rat epidermal growth factor receptor gene and its transcriptional regulation by nerve growth factor in PC12 cells.

Our previous studies have shown that treatment of PC12 cells with nerve growth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGF-induced down-regulation of the EGFR is under transcriptional control. To elucidate the molecular mechanism of this down-regulation we have cloned a 2.7-kilobase sequence from the promoter region of the rat EGFR from a rat P1 library. Six transcriptional start sites were identified by 5'-rapid amplification of cDNA ends and primer extension. Sequence analysis showed a 62% overall homology with the human EGFR promoter region. To investigate its transcription, 1.1 kilobases of the 5'-flanking sequence were fused to a luciferase reporter gene. This sequence exhibited functional promoter activity in transient transfection experiments with PC12, C6, and CV-1 cells. Treatment of PC12 cells with NGF inhibited promoter activity. By transfection of promoter deletion constructs, a silencer element was found between nucleotides -260 and -181, and TCC repeat sequences appeared to be at least partially responsible for the down-regulation of the EGFR by NGF. Supportive evidence for the relevance of this sequence was obtained from gel mobility shift assays and by transfection of TCC mutation constructs. Our results demonstrate that TCC repeat sequences are required for the down-regulation of rat EGFR by NGF in PC12 cells and may lead to the identification of the NGF-responsive transcription factors.

Animals↗

PubMed2000

Single- and multiple-dose pharmacokinetics of ziprasidone under non-fasting conditions in healthy male volunteers.

AIMS: To evaluate the pharmacokinetics and tolerability of single and multiple oral doses of ziprasidone in healthy male volunteers, and to determine the influence of ziprasidone on serum prolactin levels. METHODS: Single and multiple doses of ziprasidone were given orally (as two divided daily doses), at fixed dosages of 10 and 40 mg day(-1), and using titrated regimens of 40-80 and 40-120 mg day(-1), for 14 days. All dosages were taken immediately after food. The study adopted a randomized, double-blind, placebo-controlled design. Prolactin response, sedative properties, tolerability, and extrapyramidal symptoms were also investigated. RESULTS: Steady-state exposure to ziprasidone was attained after 1 day of dosing. Mean Cmax and AUC(0,12 h) increased with increasing dose, with apparent dose-proportionality between the 20 and 60 mg dose levels. Trough-to-peak ratios at steady state ranged from 2 to 5. Accumulation ratios for the fixed-dose regimens were 1.49 and 1.48 at the 5 and 20 mg dose levels, respectively. Ziprasidone was associated with transient prolactin elevation but levels of prolactin returned to baseline within the dosing interval at steady state. There was a marginal, transient increase in serum prolactin levels which was not dose-related at the 80 and 120 mg day(-1) doses, and which was noted to attenuate with chronic dosing. Ziprasidone was generally well tolerated. The most frequent side-effect was mild or moderate headache. A minority of patients suffered first-dose postural hypotension. Ziprasidone was also associated with a mild sedative effect that became less pronounced as treatment continued. There were no drug-related changes in electrocardiogram or clinical laboratory variables that were of clinical importance. CONCLUSIONS: Ziprasidone is characterized by a predictable pharmacokinetic profile resulting in symptoms that reflect its pharmacological action.

Adolescent↗

PubMed2000

Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression.

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed at different levels in many cell types and found overexpressed in many cancers. EGFR expression is increased or decreased in response to extracellular stimuli. We examined the effect of increased c-Jun expression on EGFR promoter activity. MATERIALS AND METHODS: We used DNAse I foot-printing analysis to determine the binding of activator protein 1 (AP-1) to the promoter region. We also used cotransfection experiments and western blotting analysis to determine the effect of AP-1 family members on EGFR expression. RESULTS: AP-1 was able to bind to at least seven sites in the EGFR promoter region. Cotransfection of MCF-7 cells with a c-Jun expression vector and the EGFR promoter reporter resulted in a 7-fold increase in promoter activity. JunB, but not c-fos, also enhanced the EGFR promoter activity. An A-Fos-dominant negative shown to inhibit Jun-dependent transactivation was able to prevent c-Jun induction of the promoter activity, but only slightly decreased the basal activity of the promoter. Furthermore, the A-Fos dominant negative was able to inhibit phorbol ester induction of the EGFR promoter. Examination of EGFR expression of MCF-7 stable cell lines that overexpress c-Jun revealed an increase in EGFR expression. Additionally, a cisplatin-resistant cell line, A2780/CP70, which has an increase in AP-1 activity compared with the parental cell line, A2780, was found to have an increase in EGFR level. CONCLUSIONS: These results indicate that AP-1 can act to increase the expression of EGFR and may play a role in upregulation of EGFR in cancer cells.

3T3 Cells↗

PubMed1999

Molecular analysis of the GCF gene identifies revisions to the cDNA and amino acid sequences(1).

GC factor (GCF) was reported as a transcriptional regulator that binds to a specific GC-rich sequence in the epidermal growth factor receptor (EGFR) gene promoter and represses its expression. In this paper, we present the data on three revisions of the cDNA sequence that lead to significant changes of the amino acid sequences of the published GCF. Firstly, 5'-rapid amplification of cDNA end (5'-RACE) analysis revealed that the 308 nucleotides of 5'-end of the previously published GCF cDNA does not exist at the 5'-end of the RACE product. Simultaneously, the correct 5'-end cDNA sequence of 31 nucleotides was identified. Secondly, the 'T' at the position 787 of the published GCF cDNA was not observed. Finally, a new sequence of 114 nucleotides was identified between the positions 851 and 852 of the published cDNA sequence. The revisions result in a GCF cDNA of 2661 nucleotides that encodes a protein of 781 amino acids, replacing the highly basic region of the amino-terminus of the published GCF with a new sequence of 147 amino acids. In this era of massive gene cloning and sequencing, this study is a warning to the biological research of recent years.

Alternative Splicing↗

PubMed1999

GC factor 2 represses platelet-derived growth factor A-chain gene transcription and is itself induced by arterial injury.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for a wide variety of cell types. The genes encoding PDGF A chain (PDGF-A) and PDGF B chain (PDGF-B) reside on separate chromosomes and are independently regulated at the level of transcription. Regulatory events underlying inducible PDGF-A expression have been the focus of much investigation. However, mechanisms that inhibit transcription of this gene are not well understood. In this study, we report the capacity of a newly cloned DNA binding factor, GC factor 2 (GCF2), to repress expression driven by the human PDGF-A promoter. 5' Deletion and transient cotransfection analysis in vascular endothelial cells revealed that GCF2 repression is mediated by a nucleotide region located in the proximal region of the PDGF-A promoter. Electrophoretic mobility shift assays demonstrate that GCF2 binds to this region in a specific and dose-dependent manner. Interestingly, the site bound by GCF2 overlaps those for specificity protein-1 (Sp1) and early growth response factor-1 (Egr-1), zinc finger transcription factors that direct basal and inducible expression of the PDGF-A gene. Gel shift experiments revealed that GCF2 competes with these factors for interaction with the PDGF-A promoter. Overexpression of GCF2 suppressed endogenous PDGF-A expression in vascular endothelial cells and smooth muscle cells. GCF2 was induced on mechanical injury of cells in culture as well as after balloon injury of the rat carotid artery wall. Time course studies revealed the sustained induction of GCF2 after injury while PDGF-A levels sharply returned to baseline. Smooth muscle cell proliferation was inhibited by GCF2, an effect reversed by the addition of exogenous PDGF-AA. These findings demonstrate negative regulation of PDGF-A expression by GCF2. This is the first report of the induction of an endogenous transcriptional repressor in the rat vessel wall.

Animals↗

PubMed1999

Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance".

BACKGROUND: Acute renal failure (ARF) leads to secondary adaptive changes that serve to protect proximal tubules from subsequent ischemic or toxic damage [so-called "acquired cytoresistance" (CR)]. A characteristic of CR is increased plasma membrane resistance to attack. Therefore, this study sought to identify potential changes in plasma membrane lipid composition in CR tubules/renal cortex and, if present, to test whether they might mechanistically contribute to the CR state. METHODS: Renal cortices/isolated tubules were obtained from CR mouse kidneys (18-hr postinduction of ischemia reperfusion, myoglobinuria, or ureteral obstruction). Their plasma membrane phospholipid/cholesterol profiles were compared with those observed in either control tissues or tissues obtained one to two hours post-renal damage (that is, prior to emergence of CR). RESULTS: Either no changes or inconsistent changes in phospholipid profiles were observed in CR tissues. Conversely, CR (vs. control) tissues demonstrated a consistent 25 to 50% increase in membrane cholesterol content. To ascertain whether cholesterol impacts tubule susceptibility to injury, its levels were reduced in proximal tubule (HK-2) cells with either (a) mevastatin, (b) a cholesterol "stripping" agent, (c) cholesterol oxidase, or (d) cholesterol esterase. Then cell susceptibility to injury [adenosine 5'-triphosphate (ATP) depletion; Fe-mediated oxidant stress] was assessed. In each instance, cholesterol reductions dramatically sensitized to superimposed injury (for example, a 2 to 3 times increase in the % of lactate dehydrogenase release). When cholesterol levels were restored to normal in CR tubules (with a "stripping" agent), an increased tubule susceptibility to injury resulted. Because cholesterol decreases membrane fluidity, the impact of a membrane-fluidizing agent (A2C) on cell injury was assessed. A2C dramatically sensitized HK-2 cells to superimposed attack. CONCLUSIONS: ARF leads to an up-regulation of proximal tubule cholesterol content. The latter may then contribute to acquired CR, possibly by stabilizing the plasma membrane via its antifluidizing effect.

Acute Kidney Injury↗

PubMed1998

Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor.

GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4. 2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility.

Amino Acid Sequence↗

PubMed1998

Interferon regulatory factor-1 is a major regulator of epidermal growth factor receptor gene expression.

Overexpression of the epidermal growth factor receptor (EGFR) occurs in many tumors and in breast cancer correlates with poor prognosis for treatment. Here, we report that interferon regulatory factor-1 (IRF-1) induces EGFR promoter activity up to 200-fold compared to 3-10-fold induction by other regulators. The region of the promoter that is required for this induction was defined using deletion mutants. In addition, we found that IRF-1 and tricostatin A, a deacetylase inhibitor, have a synergistic effect on EGFR promoter activity. This indicates that the increase in EGFR promoter activity by IRF-1 may also involve changes in chromatin structure. These results identify IRF-1 as a major regulator of EGFR gene expression.

Base Sequence↗

PubMed1998

Transcriptional down-regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells.

Treatment of PC12 cells with nerve growth factor leads to a decrease in the number of epidermal growth factor receptors on the cell membrane. The mRNA for the epidermal growth factor receptor decreases in a comparable fashion. This decrease appears due to a decrease in the transcription of the epidermal growth factor receptor gene because first, there is no difference in the stability of the epidermal growth factor receptor mRNA, second, newly transcribed epidermal growth factor receptor mRNA is decreased in nerve growth factor-differentiated cells, and third, constructs containing the promoter region of the epidermal growth factor receptor gene are transcribed much less readily in nerve growth factor-differentiated cells than in untreated cells. The decreases in mRNA are not seen in the p140(trk)-deficient variant PC12nnr5 cells nor in cells containing either dominant-negative Ras or dominant-negative Src. Treatment with nerve growth factor also increases the cellular content of GCF2, a putative transcription factor inhibitory for the transcription of the epidermal growth factor receptor gene. The increase in GCF2, like the decrease in the epidermal growth factor receptor mRNA, is not seen in PC12nnr5 cells nor in cells expressing either dominant-negative Ras or dominant-negative Src. The results suggest that nerve growth factor-induced down-regulation of the epidermal growth factor receptor is under transcriptional control, is p140(trk)-, Ras-, and Src-dependent, and may involve transcriptional repression by GCF2.

Animals↗

PubMed1998

Gamma-probe-guided resection of the sentinel lymph node in breast cancer.

Regional lymph node metastases in patients with breast cancer have fundamental staging, prognostic, and treatment implications. Classically, axillary lymph node sampling requires a dissection under general anesthesia. The concept that a primary, or sentinel, lymph node is the first node to receive drainage from a tumor has been established in patients with malignant melanomas using radiolabeled tracers and vital dyes. This study proposed two hypotheses: (1) radiolabeled sentinel lymph nodes can be identified in most patients with breast cancer, and (2) radiolabeled sentinel lymph node biopsy accurately predicts axillary lymph node metastases in those patients. Patients with operable breast cancer had Tc-99 sulphur colloid injected around their breast tumors 1-6 hours preoperatively. Patients underwent gamma probe identification of sentinel lymph nodes that were biopsied. All patients underwent axillary lymphadenectomy in conjunction with lumpectomy or mastectomy. Fifty female patients ages 26 to 90 years underwent lumpectomies with axillary dissections (40 patients) or modified radical mastectomies (10 patients). Sentinel lymph nodes were identified in 42 of 50 patients (84%). Eight patients (16%) had metastases to the axillary lymph nodes. In 7 patients, sentinel lymph nodes correctly predicted the status of the axillary nodes. There was one false negative result. A total of 550 lymph nodes were resected for an average of 11.2 nodes per patient. Sentinel lymph node scintigraphy and biopsy accurately predicted the axillary lymph node status in 41 of 42 patients (98%). Scintigraphy can identify sentinel lymph nodes in a large majority of patients. Sentinel lymph node biopsy is an accurate predictor of axillary lymphatic metastases.

Adult↗

PubMed1997

Identification of an additional p53-responsive site in the human epidermal growth factor receptor gene promotor.

Exogenously introduced wild-type and mutant p53 have recently been reported to enhance the human epidermal growth factor receptor (EGF-R) gene promoter activity in p53-deficient Saos2 osteosarcoma cells. A p53 binding site residing at position -265/-239 in the EGF-R proximal promoter has also been identified. We investigated the p53 regulation of EGF-R core promoter activity in human cell lines with varying endogenour p53 status. Wild-type and mutant p53Ala143 enhanced the EGF-R core promotor activity in cells that were either p53-deficient or contained wild-type or mutant endogenous p53. Upon further characterization of the various deletion fragments of the EGF-R promoter, we identified a wild-type p53 responsive 62 bp region residing at position -167/-105. The -167/-105 segment was responsive only to wild-type p53 but not to mutant p53Ala143 or p53His273. The -167/-105 segment of the EGF-R promotor contains one perfect and several imperfect consensus p53-binding half sites; indeed in gel shift experiments the 62 bp -167/-105 segment as well as the oligonucleotides corresponding to two p53 consensus half-sites within the 62 bp fragment, exhibited binding to p53-containing protein complexes. Thus, we have identified an additional wild-type p53 responsive site in the human EGF-R promoter. This site containing consensus p53-binding sequences resides at position -167/-105 and is proximal to recently identified p53 binding element located at position -265/-239 in the EGF-R promotor.

Base Sequence↗