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Biomedical subjects

L F Johnson

Publications and source records attributed to L F Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1991

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

Animals↗
PubMed1991

The effect of esophageal acid volume on arousals from sleep and acid clearance.

To assess the effect of different volumes of acid infused into the esophagus, seven normal volunteers were studied in the waking and sleep state. All subjects were studied for three nights in the sleep laboratory, which included complete polysomnographic monitoring and esophageal pH recording. Multiple infusions of either 5, 15, or 25 ml of 0.1 N HCL were administered each night. Similar infusions were also accomplished in the waking state. The results showed a significant (p less than 0.05) decrease in the arousal from sleep with 25-ml vs 5-ml infusions. During sleep, the latency to the first swallow was significantly (p less than .05) shorter with the 25-ml infusion when compared with that of the 5-ml infusion. While awake, the infusion volume did not affect the latency to the first swallow. The acid clearance times were not significantly altered by the different volumes infused. It is concluded that the larger volumes of acid in the esophagus create an afferent "warning" signal to the central nervous system to produce a rapid arousal from sleep along with a shortened interval to the first swallow. These responses rapidly empty the larger acid volumes from the esophagus.

Arousal↗
PubMed1990

Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding.

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.

Amino Acid Sequence↗
PubMed1990

Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame.

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.

Animals↗
PubMed1990

Gastroesophageal reflux and respiratory symptoms: is there an association? Proposed mechanisms and treatment.

Gastroesophageal reflux (GER) is a dysfunction of the distal esophagus causing movement of stomach contents into the esophagus. Patients may develop heartburn, regurgitation, dysphagia, odynophagia, and hemorrhage. Respiratory symptoms occur in 10-60 percent of patients with GER or hiatal hernia. Although there is evidence associating pulmonary symptoms and GER, causality has not been proven. The appropriate use of antireflux therapy or surgery to treat GER may consequently alleviate respiratory symptoms.

Bronchodilator Agents↗
PubMed1990

Sucralfate used as adjunctive therapy in patients with severe erosive peptic esophagitis resulting from gastroesophageal reflux.

A total of 36 patients with grade 2 or greater erosive esophagitis and an abnormal 24-h pH monitor study, were treated in a randomized, double-blind fashion to assess the efficacy of sucralfate suspension as adjunctive therapy to cimetidine for severe esophagitis secondary to gastroesophageal reflux. Treatment consisted of cimetidine, 300 mg qid and either sucralfate suspension (1 g/10 ml) or an identical placebo suspension, 10 ml after meals and 20 ml hs. Patients were treated for 12 wk unless endoscopic healing occurred earlier. Initial evaluation and monthly follow-up consisted of symptom monitoring, endoscopic evaluation and pre- and post-therapy esophageal manometry, Bernstein test, and 24-h pH monitoring. The combination of cimetidine and sucralfate suspension was superior to cimetidine alone in improving daytime heartburn symptoms (p less than 0.05) but not nighttime heartburn, dysphagia, or regurgitation. Sucralfate plus cimetidine improved the overall endoscopic outcome of esophagitis more than cimetidine alone (p less than 0.05). More patients exhibited endoscopic healing in the adjunctive sucralfate group than in the cimetidine-only group. Endoscopic healing, however, was not statistically different between groups. We conclude that sucralfate used as adjunctive therapy to cimetidine resulted in improvement of some of the symptoms of reflux, and probably increases the likelihood of complete healing of esophagitis, compared with cimetidine alone.

Cimetidine↗
PubMed1989

Efficient synthesis of mouse thymidylate synthase in Escherichia coli.

The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.

Animals↗
PubMed1989

A mouse thymidylate synthase pseudogene derived from an aberrantly processed RNA molecule.

A DNA fragment containing a mouse-thymidylate-synthase(TS) processed pseudogene was cloned and analyzed. Comparison with the sequences of the mouse TS-encoding gene (ts) and cDNA revealed that the pseudogene started at one of the normal 5' termini of TS mRNA, ended with a poly(A) tail, and was flanked by 16-nucleotide (nt) direct repeats. The region corresponding to the open reading frame was 97.3% identical to that of the cDNA. Two unusual features were observed. First, the poly(A) tail of the pseudogene was located 2 kb downstream from the normal location. Second, the final 10 nt of intron 5 were retained in the 'coding region' of the pseudogene. Therefore, it appears that the pseudogene was derived from a nonfunctional TS 'mRNA' that was aberrantly spliced and polyadenylated. Analysis of the sequence of intron 5 of the ts gene revealed the presence of an alternative 3' splice site 10 nt upstream from the normal splice site. S1-nuclease protection assays showed that about 10% of TS mRNA isolated from mouse cells was spliced at the alternative site.

Amino Acid Sequence↗
PubMed1989

Thymidylate synthase gene expression is stimulated by some (but not all) introns.

We previously described the construction of an intronless mouse thymidylate synthase (TS) minigene that has the normal 5' and 3' flanking regions of the gene linked to full length TS cDNA. Transfection of the minigene into ts- hamster V79 cells led to low level expression of normal mouse TS mRNA and protein. In the present study we analyzed the effect of introns on the expression of the TS minigene in transient transfection assays. Inclusion of introns 5 and 6 at their normal locations in the coding region led to an 8-9-fold stimulation of the level of TS and TS mRNA. Almost all of introns 5 and 6 could be deleted without diminishing the stimulatory effect. Inclusion of intron 3 also stimulated the expression of the minigene, although to a lesser extent than introns 5 and 6. However, inclusion of intron 4 had no stimulatory effect. Analysis of minigenes that contained various combinations of introns revealed that the stimulatory effects of the introns were not additive.

Animals↗
PubMed1989

Analysis of the multiple 5' and 3' termini of poly(A)+ and poly(A)-deficient thymidylate synthase mRNA in growth-stimulated mouse fibroblasts.

Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the G0/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5' and 3' termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5' or 3' termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5' termini were the same as those found in poly(A) + mRNA. However, the 3' termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.

Animals↗
PubMed1989

Cycling, a manometric phenomenon due to repetitive episodes of gastroesophageal reflux and clearance.

We studied intraesophageal pressure changes in patients with symptoms of gastroesophageal reflux and an abnormal 24-hr pH monitoring record (N = 52). Our method was simultaneous esophageal manometry and pH monitoring. We observed a three-component esophageal manometric sequence (EMS). When this sequence recurred over and over, we termed this phenomenon "cycling." We found cycling in 35% of the patients (18/52). Those with cycling had lower basal LES pressures, more acid exposure, and an increased incidence of endoscopic esophagitis. That cycling resulted from repeated reflux events and their esophageal clearance was documented by scintigraphy during simultaneous manometry and pH monitoring (N = 7 patients). Cycling was found on the routine esophageal manometry record of 25% of symptomatic patients (N = 112) with an abnormal 24-hr pH score. In conclusion, cycling represents an esophageal manometric phenomenon due to repetitive reflux events. Its recognition during esophageal manometry may denote a severe reflux diathesis.

Adult↗
PubMed1989

Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection.

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.

Animals↗
PubMed1989

The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.

Animals↗
PubMed1989

Assessment of potential for cancer control by Golden State Medical Association physicians.

To gather data on which to base the design of an intervention program to decrease cancer mortality in the black population of California, physician members of the Golden State Medical Association were surveyed to determine their current practices relating to early cancer detection and cancer prevention. Respondents' estimates of the proportions of their patients in various categories indicated that at least 60% to 70% of their patients were potential subjects for intensive early cancer detection and cancer prevention efforts. Thirty-four percent of respondents reported that they were performing early cancer detection examinations on all their patients. On the average, respondents estimated that about 70% of their patients (more than 80% of patients of primary care physicians) were receiving either routine check-ups or specific early cancer detection examinations. Patient education about various aspects of cancer and cancer prevention emerged as the most readily attainable means to reduce cancer mortality in blacks. It was most frequently mentioned both as something that physicians could do to help achieve this goal and as an outside resource physicians would find useful in their practices. Its lack was cited as the greatest barrier to early cancer detection and cancer prevention in the black population.

Adult↗
PubMed1988

Construction and expression of mouse thymidylate synthase minigenes.

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.

Animals↗
PubMed1988

Delayed processing/export of messenger RNA under conditions of reduced protein synthesis.

The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorporation of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acid-starved cells.

Animals↗
PubMed1988

Esophageal acid clearance during sleep in patients with Barrett's esophagus.

Sleep-related gastroesophageal reflux and esophageal acid clearance have been shown to be important components in the pathogenesis of reflux esophageal disease. Previous studies have suggested that patients with more severe esophagitis are distinguished by an accumulation of acid mucosal contact time during sleep. These data would suggest that patients with Barrett's esophagus should have particularly severe impairment of acid clearance, most notable during sleep. To address this issue, 16 asymptomatic healthy volunteers and 13 patients with Barrett's esophagus were studied. Acid clearance was assessed by timing the reestablishment of an esophageal pH of 4 following the infusion of 15 ml 0.1 N HCl. Sleep was polygraphically monitored in order to objectively determine sleep and waking. The results indicated that while patients with Barrett's esophagus had a marked increase in the frequency of spontaneous gastroesophageal reflux during sleep, they unexpectedly demonstrated faster acid clearance times during both waking and sleep. A greater percentage of arousal responses to acid infusion during sleep was noted in the Barrett's group. It is concluded from these results that patients with Barrett's esophagus can adequately clear acid from the distal esophagus but experience considerable acid mucosal contact through repeated episodes of spontaneous reflux during sleep.

Adult↗