Search PubMed⌕ Search

Biomedical subjects

L F Johnson

Publications and source records attributed to L F Johnson.

At least 19 recordsLinked to original sources
PubMed2006

Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene.

Thymidylate synthase (TS) is an essential enzyme that synthesizes thymidylic acid in the de novo biosynthetic pathway. Inhibiting TS enzyme activity with substrate or cofactor analogs leads to inhibition of DNA replication and cell death. For this reason, TS is an important target enzyme for cancer chemotherapeutic drugs. We describe an alternative approach to reducing cellular TS enzyme activity using short interfering RNA (siRNA) technology to lower TS mRNA levels. Plasmids that direct the synthesis of siRNAs that target nucleotides 898-916 and 965-983 (relative to the A of the translational start codon) of human TS mRNA were highly effective at reducing TS enzyme levels in transient transfection assays. Infection of HeLa cells with retroviruses that contain the effective siRNA genes led to a stable 80-95% reduction of TS enzyme and mRNA. A similar percent reduction in TS expression was observed in a cell line that overproduces TS enzyme 100-fold due to TS gene amplification. Cells that exhibited the greatest reduction in TS enzyme level grew poorly in medium that lacked thymidine. These observations suggest that siRNA approaches may provide an alternative therapeutic strategy to reduce TS enzyme levels.

Animals↗
PubMed2005

Sentinel surveillance of sexually transmitted infections in South Africa: a review.

OBJECTIVES: To review studies of sexually transmitted infection (STI) prevalence in South Africa between 1985 and 2003 in selected sentinel populations. To examine how STI prevalence varies between populations and to identify the limitations of the existing data. METHODS: Studies of the prevalence of syphilis, chancroid, granuloma inguinale, lymphogranuloma venereum, gonorrhoea, chlamydia, trichomoniasis, bacterial vaginosis, candidiasis, and herpes simplex virus type 2 (HSV-2) were considered. Results were included if they related to women attending antenatal clinics or family planning clinics, commercial sex workers, individuals in the general population (household surveys), patients with STIs, patients with genital ulcer disease (GUD), or men with urethritis. RESULTS: High STI prevalence rates have been measured, particularly in the case of HSV-2, trichomoniasis, bacterial vaginosis and candidiasis. The aetiological profile of GUD appears to be changing, with more GUD caused by HSV-2 and less caused by chancroid. The prevalence of gonorrhoea and syphilis is highest in "high risk" groups such as sex workers and attenders of STI clinics, but chlamydia and trichomoniasis prevalence levels are not significantly higher in these groups than in women attending antenatal clinics. CONCLUSIONS: The prevalence of STIs in South Africa is high, although there is extensive variability between regions. There is a need for STI prevalence data that are more nationally representative and that can be used to monitor prevalence trends more reliably.

Adolescent↗
PubMed2004

Review article: sleep and its relationship to gastro-oesophageal reflux.

Gastro-oesophageal reflux disease (GERD) is among the most common gastrointestinal conditions in the USA. For most symptomatic patients, reflux events occur during both daytime and night-time hours. Whereas daytime reflux events tend to be frequent but brief, reflux events that occur during sleep are comparatively less frequent but significantly longer. Longer oesophageal acid-clearance and acid-mucosal contact times during sleep are at least partly due to several physiological changes associated with sleep, including dramatic declines in saliva production and frequency of swallowing, decreased conscious perception of heartburn and consequent arousal and clearance behaviours, and slower gastric emptying. Obstructive sleep apnea syndrome and obesity seem to predispose some patients to nocturnal GERD, and the presence of either of these conditions may help to identify patients with symptoms consistent with GERD. Recognition and treatment of night-time GERD are important because it can be associated with decreased quality of life (including sleep disruption) and increased risk of serious oesophageal and respiratory complications.

Circadian Rhythm↗
PubMed2001

The anticytomegaloviral activity of raltitrexed is abrogated in quiescent mouse fibroblasts that overexpress thymidylate synthase.

Cytomegalovirus (CMV) replication in non-proliferating cells requires the coordinated expression of the host enzymes responsible for deoxyribonucleotide synthesis. Thymidylate synthase (TS) is an essential cellular enzyme that catalyzes de novo synthesis of thymidylic acid (dTMP). In this report we show that murine CMV (MCMV) replication and DNA synthesis are inhibited in quiescent 3T6 fibroblasts by raltitrexed, a quinazoline-based folate analog that specifically inhibits TS. This antiviral activity was abrogated in LU3-7 cells, a 3T6 derivative that overproduces TS by about 50-fold. These observations indicate that the anticytomegaloviral activity of raltitrexed is associated with TS inhibition and suggest that cellular TS activity is required for efficient CMV replication in quiescent cells.

Animals↗
PubMed2001

Involvement of level I neck lymph nodes in advanced squamous carcinoma of the larynx.

This study was performed to evaluate the incidence of metastasis at level I in patients with squamous laryngeal cancer. One hundred consecutive patients with squamous carcinoma of the larynx were submitted to surgical treatment including radical neck dissection. The tumor stage was T3 or T4, and the neck stage was N1-N2c. Lymph node metastases were pathologically confirmed in 80 patients. Metastases were concentrated within level II in 59% of cases, level III in 17% of cases, level IV in 11% of cases, and level V in 6% of cases. Only 2 patients (2%) had detectable tumors in the lymph nodes of the submandibular triangle (level IB). This study shows that patients with laryngeal cancer rarely present metastases at the submandibular triangle, even in advanced local disease with cervical metastasis staged as N1 to N2c. Therefore, dissection of the submandibular triangle is indicated only in the presence of clinical, radiographic, or cytologic evidence of metastatic disease at level I.

Adult↗
PubMed2001

Closing the gap.

Explore the source record for details and available documents.

Foot Injuries↗
PubMed2000

Inhibition of the mammalian transcription factor LSF induces S-phase-dependent apoptosis by downregulating thymidylate synthase expression.

The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.

Amino Acid Sequence↗
PubMed2000

Transcriptional control elements of the rat thymidylate synthase promoter: evolutionary conservation of regulatory features.

The sequence elements that are important for the transcription and regulation of the rat thymidylate synthase (TS) gene were analyzed. The rat TS promoter lacks a TATA box and directs transcriptional initiation at multiple sites between 60 and 20 nt upstream of the AUG translational start codon. Promoter deletion analyses showed that the region between -100 and -42 nt relative to the AUG codon was both necessary and sufficient for high level promoter activity and was designated the essential promoter region. The essential region also had bidirectional promoter activity. Site-directed mutagenesis revealed that four elements were especially important for promoter activity. These include Ets motifs at -85 and -50, an Sp1 motif at -80, and an LSF motif that overlapped the upstream Ets and Sp1 motifs. Inactivation of E2F motifs that are upstream and downstream of the essential promoter region had no measurable effect on promoter activity in transient transfection assays. The rat TS promoter region directed S-phase-specific expression of a stably transfected minigene if a spliceable intron was included in the transcribed region. When the intron was deleted or the E2F motifs were inactivated, expression of the minigene changed very little during the G1 to S transition.

Animals↗
PubMed2000

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter.

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

Animals↗
PubMed2000

Proximal migration of esophageal acid perfusions during waking and sleep.

OBJECTIVE: Proximal acid migration resulting from gastroesophageal reflux has been implicated in aerodigestive complaints and disorders. This study was designed to investigate the effects of acid volume, posture, and sleep on proximal esophageal acid migration (drop in pH to <4.0). METHODS: The study was performed in 15 healthy adults. A distal esophageal acid perfusion technique to simulate gastroesophageal reflux was used. Esophageal acid perfusions of 1 ml and 3 ml were accomplished at a site 5 cm above the proximal border of the lower esophageal sphincter in the upright and supine positions during waking, and during polysomnographically monitored sleep. Esophageal pH was recorded by two sensors located in the mid- and proximal esophagus at 10 and 15 cm above the lower esophageal sphincter. RESULTS: Acid volume clearly increased the incidence of migration to the mid and proximal sensors during both waking and sleep, and also significantly increased acid clearance time. Posture failed to significantly affect the incidence of acid migration and acid clearance. Sleep clearly enhanced migration to the proximal pH sensor of even those perfusions as small as 1 ml. For instance, 40% of 1 ml perfusions during sleep migrated to the proximal sensor compared with <1% during waking. Acid clearance times were significantly longer during sleep as measured by the mid- and proximal esophageal pH sensors. CONCLUSIONS: In healthy individuals, volume enhances the likelihood of migration to both mid- and proximal esophagus, and significantly prolongs clearance time in the waking state. Posture appears to be a less significant parameter with regard to both the incidence of acid migration and acid clearance. Sleep is a significant risk factor for acid migration to the proximal esophagus for even minute volumes, and markedly prolongs acid clearance.

Adult↗
PubMed2000

Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication.

Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.

3T3 Cells↗
PubMed2000

Intramural tracking: a feature of esophageal intramural pseudodiverticulosis.

OBJECTIVE: Our purpose was to determine the frequency of intramural tracking in patients with esophageal intramural pseudodiverticulosis and to characterize the morphologic features of this finding on barium studies. MATERIALS AND METHODS: A review of radiology files at two institutions revealed 30 cases of esophageal intramural pseudodiverticulosis diagnosed at esophagography. In all cases, the radiographs were reviewed retrospectively to determine the frequency and morphologic features of intramural tracking in these patients. The number and distribution of pseudodiverticula and the presence or absence of strictures or esophagitis were also noted. RESULTS: Fifteen (50%) of 30 patients with esophageal intramural pseudodiverticulosis had intramural tracking on esophagography. The tracks had an average length of 1.2 cm (length range, 0.3-7 cm) and an average width of 1.6 mm (width range, 1-4 mm). The pseudodiverticula were more numerous and had a more diffuse distribution in patients with tracking than in patients without tracking. Although patients with and without tracking had a similar frequency of strictures and esophagitis, patients with tracking were more likely to have strictures involving the upper or mid esophagus, whereas patients without tracking were more likely to have strictures in the distal esophagus. These findings indicate that intramural tracking is more likely to occur in patients with the diffuse form of esophageal intramural pseudodiverticulosis. CONCLUSION: Intramural tracking was detected on esophagography in 50% of patients with esophageal intramural pseudodiverticulosis, so this type of tracking is a more common radiographic finding than has previously been recognized. Although intramural tracking has little or no known clinical significance, it is important to be aware of this finding so that it is not mistaken for a large flat ulcer in the esophagus or for an extramural collection associated with esophageal peridiverticulitis.

Adolescent↗
PubMed2000

Unseen dangers.

Explore the source record for details and available documents.

Facility Regulation and Control↗