Biomedical subjects

M Chang

Publications and source records attributed to M Chang.

At least 109 records · Page 6Linked to original sources

PubMed1995

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

Adult↗

PubMed1995

In vivo imaging of brain incorporation of fatty acids and of 2-deoxy-D-glucose demonstrates functional and structural neuroplastic effects of chronic unilateral visual deprivation in rats.

Regional cerebral 'incorporation coefficients' k* of each of 3 labeled long-chain fatty acids -[9,10-3H]palmitate ([3H]PA), [1-14C]arachidonate ([14C]AA) and [1-14C]docosahexaenoate ([14C]DHA)-were measured using quantitative autoradiography in 11 bilateral brain visual areas of 3.5-month-old awake, hooded, Long-Evans rats, and were compared with regional cerebral metabolic rates for glucose (rCMRglc). The rats, which had undergone unilateral orbital enucleation at 15 days of age, were studied either in the dark with eyelids of the intact eye sutured, or when stimulated in a light box with the intact eye open. rCMRglc did not differ between homologous contralateral and ipsilateral visual areas in the dark or during stimulation, but was elevated bilaterally by 25% or more in many visual areas during stimulation compared with dark. Contralateral compared with ipsilateral k* was lower for each fatty acid tracer in superficial gray of the superior colliculus (in dark and during stimulation) and dorsal nucleus of lateral geniculate body (during stimulation). In the dark, k* for [3H]PA was correlated significantly with rCMRglc for the 22 visual areas studied, whereas during stimulation k* for [14C]AA was correlated with rCMRglc. These results suggest that central neuroplastic changes following chronic unilateral enucleation are accompanied by reduced incorporation of [3H]PA, [14C]AA and [14C]DHA into contralateral brain ares that normally receive crossed retinofugal fibers, and by symmetry of rCMRglc in the dark but increased bilateral symmetrical responsiveness of rCMRglc to visual stimulation of the intact eye.

Animals↗

PubMed1995

Central pontine myelinolysis after liver transplantation: a case report.

A patient development deteriorating mental status, quadriparesis, and severe pseudobulbar palsy with the inability to speak or swallow following orthotopic liver transplantation (OLT). Subsequently, abnormalities were found in the pons on MRI that were consistent with central pontine myelinolysis (CPM). Marked recovery occurred following transfer to the rehabilitation medicine service. Seven months following development of CPM, a mild dysarthria has persisted, but full ambulation has returned. Although no significant fluctuations in serum sodium were seen perioperatively, multiple risk factors associated with the development of CPM were present, including end-stage liver disease, a history of alcohol abuse, malnutrition, hypoxia, and use of cyclosporin medication postoperatively. This case demonstrates that the development of CPM may occur following OLT despite meticulous attention to serum sodium concentrations. We conclude that CPM is multifactorial in nature. There can be a great variation in its clinical course.

Adult↗

PubMed1995

Automated analytical systems for drug development studies. 3. Multivessel dissolution testing system based on microdialysis sampling.

An automated system consisting of a six-vessel dissolution apparatus, microdialysis sampling, STT E6 multiposition switching valve and a liquid chromatograph was assembled to measure dissolution profiles of immediate and sustained-release tablets. A DL-5 microdialysis loop probe (BAS, Inc.) was immersed in each dissolution vessel and perfused with a suitable medium for sampling. The dialystate from each vessel was injected sequentially onto an on-line liquid chromatography (LC) system for automated analysis. The STT E6 multiposition switching valve was used to sample up to six vessels simultaneously. After addressing issues related to sample carry-over and between-probe variability, the automated system was used in a reproducible manner (RSD < 3%) to measure the dissolution of immediate-release acetaminophen tablets and Accutrim (containing 75 mg phenylpropanolamine HCl) 16 h Precision Release tablets. An uneven injection time sequence was used to monitor three acetaminophen tablets per dissolution run using the automated system and each vessel was sampled about every 6.5 min. However, with Accutrim 16 h Precision Release tablets, a longer sampling interval (10 min) was used, the six tablets could be tested in each dissolution run. The dissolution profiles of acetaminophen and Accutrim tablets measured using the automated multivessel dissolution system compared well with manual and automated single-vessel dissolution systems.

Acetaminophen↗

PubMed1995

Specific activity of brain palmitoyl-CoA pool provides rates of incorporation of palmitate in brain phospholipids in awake rats.

In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2-10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with > 50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6-1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by approximately 60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.

Animals↗

PubMed1995

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype.

A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1995

Interphenotype differences in disposition and effect on gastrin levels of omeprazole--suitability of omeprazole as a probe for CYP2C19.

1. Fourteen healthy Swedish Caucasian subjects were given 20 mg of omeprazole orally each morning for 8 days. The subjects included five poor metabolisers (PM) of S-mephenytoin, four heterozygous extensive metabolisers (hetEM) and five subjects with a very rapid metabolism (rapidEM). 2. After the first dose, the relative mean areas under the plasma concentration vs time curve (AUC) of omeprazole in rapidEM, hetEM and PM were 1:3.7:20 (all different, P < 0.001). A similar relation was seen in the AUC(0,10 h) of the sulphone metabolite (1:3:12). Concentrations of hydroxyomeprazole were higher in EM than in PM confirming that the hydroxy, but not the sulphone metabolite, is formed by the S-mephenytoin hydroxylase (CYP2C19). After 8 days of treatment, the differences between groups were similar. 3. After both the first and the eighth doses, the omeprazole/hydroxyomeprazole plasma concentration ratio, determined 3 h after drug intake, correlated with the mephenytoin S/R ratio (rs = 0.94; P < 0.001; n = 14) suggesting that omeprazole might be used to phenotype for CYP2C19. 4. After the first dose of omeprazole, there was no difference in the AUC(0,10 h) of plasma gastrin between the three groups. From the first to the eighth dose, the AUC(0,10) of gastrin increased significantly in both hetEM and PM, while there was no change in the rapidEM. After the eighth dose, the AUC(0,10) of gastrin correlated significantly with the AUC of omeprazole in plasma (rs = 0.79; P < 0.01; n = 13).

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1995

Map dynamic ranges versus duration of hearing loss in cochlear implantees.

In order to assess the relationship of factors such as age, cause of deafness, use of hearing aids, duration of hearing loss, and age at onset of hearing loss, with the size of map dynamic ranges (DRs), 34 postlingually and 8 prelingually deaf users of the Nucleus cochlear implant system were analyzed according to the sizes of map DRs. Lack of auditory stimulation for a longer period than 10 years might be one of the most important factors associated with a reduced map DR. Use of hearing aids was associated with larger map DRs. Small map DRs were found across a range of age, age at onset, and duration of hearing loss. Cause of deafness did not appear to be related to map DRs.

Adolescent↗

PubMed1995

Coxiella burnetii in polymorphic lymphocytes in tissue and blood of patients with polymorphic reticulosis.

Coxiella burnetii is a well-known causative agent of granulomatous inflammation and an inducer of morphological changes and transformation of human B lymphocytes in vitro. An association of the organism with polymorphic reticulosis (PMR), a malignant granulomatous inflammation characterized by polymorphic lymphocytes, was examined. The infection of C. burnetii was demonstrated in all cases tested, especially in polymorphic lymphocytes. Also the presence of morphologically transformed peripheral blood lymphocytes (PBLs) infected with C. burnetii was demonstrated. In cultures of blood lymphocytes, C. burnetii-infected polymorphic cells identified as B cells became immortalized in vitro. These findings implicate the role of C. burnetii in the process of PMR.

Adult↗

PubMed1994

Regulation of interleukin-11 protein and mRNA expression in neonatal and adult fibroblasts and endothelial cells.

Interleukin-11 (IL-11), a newly-identified cytokine produced by stromal cells, elevates platelet counts in neonatal rats in vivo and synergizes in vitro with IL-3 in supporting murine megakaryocyte colony formation and stimulating hematopoietic stem cells. Megakaryocytopoiesis is also enhanced by other colony-stimulating factors (CSFs), including IL-3, IL-6, and Steel factor (SLF). Dysregulation of neonatal thrombopoiesis predisposes newborns to develop thrombocytopenia during sepsis, despite increased circulating pools of committed thrombopoietic progenitors in newborn cord blood compared with adult. We previously reported reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and IL-3 from stimulated cord mononuclear cells, but increased expression of SLF in human umbilical vein endothelial cells (HUVEC). Therefore, we hypothesized that IL-3, IL-6, and SLF might modulate megakaryocytopoiesis by inducing IL-11 expression, and newborns might express altered levels of IL-11 mRNA expression during activated conditions, contributing to the difference in circulating colony-forming unit-megakaryocyte (CFU-Meg) cord and adult blood. Phorbol myristate acetate (PMA) induced a twofold greater increase in IL-11 mRNA expression in neonatal fibroblasts (NFb) compared with adult fibroblasts (AFb), and a 3.6-fold greater increase in HUVEC than human adult aorta endothelial cells (HAEC) by Northern blot analysis. PMA also induced a threefold greater increase in IL-11 protein production in NFb than AFb. Physiologic agonists IL-1 alpha, transforming growth factor-beta 1 (TGF-beta 1), and TGF-beta 2 triggered upregulation of IL-11 mRNA expression in both NFb and AFb. However, IL-3, IL-6, PIXY321 (a GM-CSF-IL-3 fusion protein), and SLF failed to upregulate IL-11 mRNA expression from the basal level, while macrophage-colony stimulating factor (M-CSF) mRNA was significantly induced. These data suggest that the hematopoietic effect of IL-6, SLF, and IL-3 on megakaryocytopoiesis is probably not mediated by secondary IL-11 mRNA expression. Similarly, inflammatory agonists IL-1 beta, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone did not upregulate IL-11 expression from the basal level in endothelial cells, whereas intracellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 were strongly induced. Minimal basal IL-11 expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in NFb, AFb, HUVEC and HAEC. The quantitative RT-PCR assay also verified that IL-1 beta and TNF-alpha-stimulated HUVEC and HAEC, and IL-3- and IL-6-stimulated NFb and AFb only expressed minimal levels of IL-11 mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1994

Transforming growth factor-beta 1, macrophage inflammatory protein-1 alpha, and interleukin-8 gene expression is lower in stimulated human neonatal compared with adult mononuclear cells.

Hematopoiesis is developmentally immature in the newborn compared with the adult. Diminished gene expression of several positive hematopoietic regulators has been observed in activated cord compared with adult peripheral blood mononuclear cells (MNC; Cairo et al. Pediatr Res, 30:362, 1991 and Cairo et al, Pediatr Res, 31:574, 1992). However, altered expression of negative hematopoietic regulators during states of increased demand may also contribute to the pathogenesis of newborn dyshematopoiesis. To test this hypothesis, we measured protein levels of transforming growth factor-beta 1 (TGF-beta 1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the conditioned media of human umbilical cord and adult MNC using specific enzyme-linked immunosorbent assays. There was significantly less TGF-beta 1 in culture supernatants of cord versus adult MNC after 24, 72, and 120 hours of stimulation (P < .05), and significantly less MIP-1 alpha in cord versus adult supernatants after 72 hours and 120 hours of stimulation (P < .01). We then examined the mRNA expression of the negative regulators TGF-beta 1, MIP-1 alpha, and interleukin-8 (IL-8) in cord and adult MNC using Northern blot hybridization followed by quantitative densitometry. Cord MNC expressed significantly less TGF-beta 1 mRNA than adult MNC 6 hours and 72 hours after stimulation (P < .001). Cord MNC expressed significantly less MIP-1 alpha mRNA than adult MNC 6 hours (P < .01), 24 hours (P < .001), and 72 hours after stimulation (P < .001). Cord MNC also expressed significantly less IL-8 mRNA than adult MNC 6 hours after stimulation (P < .001). Therefore, decreased mRNA accumulation appears to coincide with reduced cytokine expression in the activated cord MNC. There were no significant differences in the transcription rates determined by nuclear run-on assay of either the TGF-beta 1 or MIP-1 alpha gene in cord versus adult MNC after 6 hours of stimulation, suggesting that the reduced TGF-beta 1 and MIP-1 alpha mRNA in activated cord MNC may be secondary to alteration in posttranscriptional regulation. The present results, together with those of our previous studies, suggest that the altered expression of both positive and negative hematopoietic regulators may be involved in the immaturity of host defense in human neonates.

Adult↗

PubMed1994

Evidence against DNA polymerase beta as a candidate gene for Werner syndrome.

Werner syndrome (WS) is a rare autosomal recessive disorder of humans characterized by the premature onset and accelerated rate of development of several major age-related disorders. An aberration in DNA replication or repair is suggested by the evidence of genome instability. Since the structural gene for DNA polymerase beta maps within the region of the WS mutation on the short arm of chromosome 8 and is involved in both DNA repair and DNA replication, we evaluated its candidacy as the WS gene. Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase beta gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.

Cell Line↗

PubMed1994

Automated analytical systems for drug development studies. II--A system for dissolution testing.

Microdialysis is a non-equilibrium dynamic sampling method in which the analytes diffuse across a semipermeable membrane due to a concentration gradient and are carried away by the constantly pumping perfusion medium for on-line analysis. A BAS, Inc. microinfusion pump/injector and an on-line LC system were interfaced with a dissolution apparatus to automate dissolution testing of tablets. A DL-5 microdialysis loop probe was suspended in the dissolution medium for sampling. The automated system was used reproducibly (RSD < 2%) to measure the dissolution of acetaminophen and Sulfatrim tablets. Drug recovery from the microdialysis probe was a function of the perfusion rate at constant temperature. However, microdialysis recovery was independent of drug concentration over the linear ranges of the assays for the analytes of interest. Dissolution profiles determined by microdialysis sampling were compared with manual sampling. Identical profiles were obtained for acetaminophen tablets in water at 37 degrees C and 50 rpm by both sampling methods. Dissolution of Sulfatrim tablets was determined in 0.1 M hydrochloride acid at 37 degrees C and 75 rpm. Microdialysis sampling permitted the use of a specially designed perfusion medium to buffer the acidic samples before injecting onto the LC column. Dissolution profiles of sulphamethoxazole were comparable for both sampling methods; however, microdialysis sampling indicated slightly higher release of trimethoprim from the Sulfatrim tablets, which was attributed to release of adsorbed drug from the connecting tubing.

Acetaminophen↗

PubMed1994

Antiproliferative activity to vascular smooth muscle cells and receptor binding of heparin-mimicking polyaromatic anionic compounds.

Proliferation of bovine aortic smooth muscle cells (SMCs) induced by thrombin, basic fibroblast growth factor, or serum is inhibited by anionic, nonsulfated aromatic compounds that mimic many of the effects of heparin. Among these compounds are aurintricarboxylic acid (ATA) and a newly synthesized polymer of 4-hydroxyphenoxy acetic acid (compound RG-13577). Iodinated- or 14C-labeled compound RG-13577 binds to cultured SMCs in a highly specific and saturable manner. Scatchard analysis of the binding data revealed the presence of an estimated 1 x 10(7) binding sites per cell with an apparent dissociation constant of 3 x 10(-6) mol/L. Binding of radiolabeled RG-13577 was efficiently competed for by related aromatic anionic compounds and by apolipoprotein E, but not by heparin, heparan sulfate, suramin, or various purified growth factors and extracellular matrix proteins. Receptor cross-linking of SMC-bound 125I-RG-13577 revealed a single species of high M(r) (approximately 280 kD) cell surface receptors detected in the absence but not the presence of excess unlabeled compound RG-13577. Binding was susceptible to downregulation and restoration of receptor levels in a manner similar to that of hormone and growth factor receptors. We suggest that the antiproliferative activity of compound RG-13577 and related compounds is initiated by binding to specific growth-inhibiting cell surface receptors. Heparin-mimicking compounds may be applied to inhibit SMC proliferation associated with atherosclerosis and restenosis.

Animals↗

PubMed1994

Evaluation of standard and active compression-decompression CPR in an acute human model of ventricular fibrillation.

BACKGROUND: The mechanisms that underlie cardiopulmonary resuscitation (CPR) in humans remain controversial and difficult to study. This report describes a new human model to evaluate CPR during the first 1 to 2 minutes after the onset of ventricular fibrillation (VF). With this model, standard CPR was compared with active compression-decompression (ACD) CPR, a method that uses a handheld suction device to actively compress and actively decompress the chest. METHODS AND RESULTS: During routine inductions of VF as part of a transvenous lead cardioverter/defibrillator implantation procedure, CPR was performed in 21 patients if the first defibrillation shock failed and until a successful rescue shock was delivered. Compressions during CPR were performed according to American Heart Association guidelines. For ACD CPR, decompression was performed with up to -30 lbs. Radial arterial and right atrial pressures were measured in all patients. Esophageal pressures, intratracheal pressures, or minute ventilation was measured in the last 13 patients. Application of both CPR techniques increased arterial and right atrial pressures. The mean coronary perfusion pressure was increased throughout the entire CPR cycle with ACD CPR (compression, 21.5 +/- 9.0 mm Hg; decompression, 21.9 +/- 8.7 mm Hg) compared with standard CPR (compression, 17.9 +/- 8.2 mm Hg; decompression, 18.5 +/- 6.9 mm Hg; P < .02 and P < .02, respectively). Ventilation per compression-decompression cycle was 97.3 +/- 65.6 mL with standard CPR and 168.4 +/- 68.6 mL with ACD CPR (n = 7, P < .001). Negative inspiratory pressure was -0.8 +/- 4.8 mm Hg with standard CPR and -11.4 +/- 6.3 mm Hg with ACD CPR (n = 6, P < .04). CONCLUSIONS: Patients undergoing multiple inductions of VF during cardioverter/defibrillator implantation with transvenous leads provide a well-controlled and reproducible model to study the mechanisms of CPR. Using this model, ACD CPR significantly increased arterial blood pressure, coronary perfusion pressure, minute ventilation, and negative inspiratory pressure compared with standard CPR.

Acute Disease↗

PubMed1994

Deletion mapping of chromosome 8p in colorectal carcinoma and dysplasia arising in ulcerative colitis, prostatic carcinoma, and malignant fibrous histiocytomas.

Short tandem repeat polymorphism markers on the short arm of chromosome 8 were used to search for loss of heterozygosity (LOH) in colorectal carcinoma and dysplasia complicating ulcerative colitis, in prostatic carcinoma, and in malignant fibrous histiocytoma (MFH). Fifty percent of prostatic carcinomas (13/26), 44% of carcinomas or dysplasias arising in ulcerative colitis (7/16), and 30% (4/12) of MFH cases showed LOH for markers on 8p. Detailed mapping demonstrated variability in the size of the chromosomal region showing LOH; however, the data suggest a common 30-centimorgan region of LOH on chromosome 8p between the LPL locus and pter in colorectal and prostatic cancers. In addition, LOH was observed on 8p in both high-grade and low-grade dysplasia in ulcerative colitis, indicating that LOH on 8p may occur at an early stage of neoplastic development in this disorder. In contrast, MFH cases exhibited LOH for marker D8S87, which has been identified as being near the putative Werner's syndrome locus. These results suggest that a tumor suppressor gene, located on the distal portion of chromosome 8p, exists in common for prostatic and colorectal carcinomas, and a second tumor suppressor gene may exist linked to the Werner's syndrome locus.

Chromosome Deletion↗

PubMed1993

Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase alpha-subunit gene.

We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.

Base Sequence↗

PubMed1993

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.

Amino Acid Sequence↗