Simplified filter paper sandwich blot provides rapid, background-free northern blots.
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Blotting, Northern↗
Biomedical subjects
R W Jones
Publications and source records attributed to R W Jones.
Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene.
Erythropoietin, the major hormone controlling red-cell production, is regulated in part through oxygen-dependent changes in the rate of transcription of its gene. Using transient transfection in HepG2 cells, we have defined a DNA sequence, located 120 base pairs 3' to the poly(A)-addition site of the mouse erythropoietin gene, that confers oxygen-regulated expression on a variety of heterologous promoters. The sequence has the typical features of a eukaryotic enhancer. Approximately 70 base pairs are necessary for full activity, but reiteration restores activity to shorter inactive sequences. This enhancer operates in HepG2 and Hep3B cells, but not in Chinese hamster ovary cells or mouse erythroleukemia cells, and responds to cobalt but not to cyanide or 2-deoxyglucose, thus reflecting the physiological control of erythropoietin production accurately.
Animals↗
Reflections on carcinoma in situ.
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Carcinoma in Situ↗
1991 cervical screening recommendations: a working group report.
The Department of Health and the Cancer Society invited a working group to review the 1985 recommendations on cervical screening. Minor modifications have been made to the earlier recommendations in the light of more information about the effectiveness of different screening policies, and in the expectation that comprehensive cytology registers to ensure recall and follow up of abnormalities will be in place shortly. All women from the age of 20 up to 70 years should be offered cervical screening every three years. Any woman who has never had sexual intercourse or who has had a hysterectomy with complete removal of the cervical epithelium for a benign condition need not be screened. Women should have a second smear within one year if they have never had a smear before or if more than five years have passed since their last smear. But recall through the register should not be more frequently than three yearly for women with a history of normal smears.
Adult↗
Possible etiologic heterogeneity of vulvar intraepithelial neoplasia. A correlation of pathologic characteristics with human papillomavirus detection by in situ hybridization and polymerase chain reaction.
A correlated histopathologic and molecular virologic study of 30 cases of vulvar intraepithelial neoplasia Grade 3 (VIN 3) and six associated invasive vulvar carcinomas was performed. Paraffin sections were examined for human papillomavirus (HPV) types 6, 11, 16, and 18 by in situ hybridization for viral transcripts and by polymerase chain reaction (PCR) for amplification of HPV and of the beta-globin gene. Vulvar intraepithelial neoplasia Grade 3 was histologically subclassified into warty (bowenoid) (20 cases) and basaloid (undifferentiated) (ten cases) types. Warty VIN characteristically was composed of squamous cells displaying abnormal proliferation and maturation and an undulating or spiked surface creating a "condylomatous" appearance whereas basaloid VIN had a smooth surface and was composed of undifferentiated basaloid cells resembling carcinoma in situ of the cervix. Human papillomavirus-16 was the only type detected in 16 of 30 VIN 3 and in five of six invasive carcinomas. The HPV-positive women were younger than HPV-negative women (mean age at diagnosis, 49 versus 60 years), their lesions more frequently demonstrated koilocytotic atypia (94% versus 43%), and they were more likely to have warty compared with basaloid VIN lesions (65% versus 30%). These findings suggest that there are at least two different types of VIN which have differing clinical, pathologic, and viral profiles.
Adult↗
The effect of age on the pharmacokinetics of pentisomide.
The effects of age on the pharmacokinetics of pentisomide (CM7857), an orally effective antiarrhythmic agent, were studied in two groups of volunteers. Sixteen young volunteers (mean age 26.4 years) and 10 elderly volunteers (mean age 67.8 years) received a single 200 mg oral dose of pentisomide. Mean AUC was larger and terminal elimination half-life longer in the elderly subjects, due to a decrease in total plasma clearance of pentisomide in the elderly. This decrease was due to a reduction in renal clearance of the drug which was paralleled by a significantly lower creatinine clearance in the elderly subjects. Dosage reduction, or a reduced frequency of dosing of pentisomide would be necessary in the elderly or those with impaired renal function.
Administration, Oral↗
Drosophila genome project: one-hit coverage in yeast artificial chromosomes.
We present a strategy for assembling a physical map of the genome of Drosophila melanogaster based on yeast artificial chromosomes (YACs). In this paper we report 500 YACs containing inserts of Drosophila DNA averaging 200 kb that have been assigned positions on the physical map by means of in situ hybridization with salivary gland chromosomes. The cloned DNA fragments have randomly sheared ends (DY clones) or ends generated by partial digestion with either NotI (N clones) or EcoRI (E clones). Relative to the euchromatic portion of the genome, the size distribution and genomic positions of the clones reveal no significant bias in the completeness or randomness of genome coverage. The 500 mapped euchromatic clones contain an aggregate of approximately 100 million base pairs of DNA, which is approximately one genome equivalent of Drosophila euchromatin.
Animals↗
The comparative pharmacokinetics of acemetacin in young subjects and elderly patients.
Single and multiple dose pharmacokinetics of acemetacin in 10 young healthy subjects and 10 elderly patients with osteoarthritis were studied. Peak plasma concentrations of acemetacin and its metabolite, indomethacin, were found between 2.4 and 4 h after an oral dose of the drug. After dosing to steady state (7 days), the mean plasma elimination half-life for acemetacin was 1.1 h and 1.0 h, and for indomethacin 7.1 h and 7.2 h in the young and elderly groups respectively. Statistical analysis of tmax, AUC, plasma t1/2 and residual drug concentrations for acemetacin or indomethacin revealed no significant differences (P greater than 0.05) between young subjects and elderly patients after acute or chronic dosing. The results suggest that drug accumulation did not occur in the elderly subjects over the time period studied and that, on pharmacokinetic grounds, dose adjustment in the elderly is unlikely to be required.
Adult↗
Effects of NMDA modulation in scopolamine dementia.
D-Cycloserine is a partial agonist at the strychnine-insensitive neuronal glycine receptor and positively modulates the N-methyl-D-aspartate (NMDA) excitatory amino acid receptor. NMDA receptors appear to be important in learning and memory, and D-cycloserine facilitates learning in rats. In man, central cholinergic blockade due to scopolamine administration impairs attention, information processing, and memory for new information, the latter secondary memory impairments resembling those shown in patients with Alzheimer's disease (AD). D-Cycloserine has been studied for its ability to counteract the cognitive decrements produced by scopolamine in young and elderly healthy volunteers. D-Cycloserine specifically antagonized the memory impairments produced by scopolamine. These findings provide evidence of NMDA receptor involvement in human memory and suggest a novel mechanism for alleviation of memory loss associated with aging and dementia.
Adolescent↗
Recombinant porcine somatotropin alters performance and carcass characteristics of heavyweight swine and swine fed alternative feedstuffs.
The efficacy of recombinantly derived porcine somatotropin (rpST) in improving the performance and carcass characteristics of heavyweight finishing pigs was determined. In Study 1, 36 pigs were killed for determination of initial carcass composition at 102 kg, and 36 pigs each were given 0, 1.5, 3, 6, or 9 mg of rpST/d from 102 to 136 kg live weight. Corn-soybean meal diets fed contained 19.8% CP and greater than 1% lysine. Study 2 tested the effects of 3 mg/d on pigs for 4 wk from 84 kg BW fed a corn-soybean meal or a triticale-barley-peas diet. Performance variables (ADG, average daily feed intake [ADFI], and feed efficiency [FE]) were measured weekly. Treatment with rpST increased (P less than .01) ADG and FE and decreased (P less than .01) ADFI in both studies. In Study 1, leaf fat, backfat thickness, belly thickness, and carcass fat were all decreased (P less than .01) linearly by rpST. Loin eye area (LEA), total trimmed lean, and total protein content were increased quadratically (P less than .01). At the 3-mg dose, ADFI, ADG, and FE were 87, 130, and 137% of control, and LEA, backfat, total protein gain, and total fat gain were 107, 80, 136, and 52% of control. Loin eye area was not increased (P greater than .05) in the pigs in Study 2; however, backfat thickness was reduced 16% by rpST (P less than .01). The effects of rpST were the same on both dietary regimens (P greater than .05). These studies demonstrate the effectiveness of rpST in increasing ADG and carcass leanness and improving FE in heavyweight pigs and in pigs fed alternative feedstuffs.
Adipose Tissue↗
Transient infrared transmission spectroscopy.
Transient infrared transmission spectroscopy is a new method that can acquire analytically useful transmission spectra from moving, optically thick solids. No sample preparation is required. The spectra are of sufficient quality for accurate quantitative compositional analysis. The method works by the creation of a thin, short-lived, chilled layer at the sample surface. Blackbody-like thermal emission from the bulk of the sample is selectively absorbed as it passes through the chilled layer, so the transmission spectrum of the layer is superimposed on the observed thermal emission. Spectra of polycarbonate, beeswax, and copolymers of methyl and butyl methacrylate are presented. Compositional analysis of the methacrylate copolymers with a standard error of prediction of only 0.87 mol % is demonstrated.
Chemistry Techniques, Analytical↗
Oxygen-dependent modulation of erythropoietin mRNA levels in isolated rat kidneys studied by RNase protection.
Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.
Animals↗
Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement.
Barley stripe mosaic hordeivirus (BSMV) has a tripartite positive-sense RNA genome which encodes seven major polypeptides. Infectious in vitro transcripts derived from full-length wild-type and mutant cDNA clones have been used to investigate the contribution made by various BSMV gene products to viral RNA replication and systemic movement. We show that whereas all three of the BSMV RNA components are required for plant infection, RNAs alpha and gamma can replicate together in barley protoplasts, and therefore RNA beta must encode functions required for systemic invasion of plants. The alpha a and gamma a proteins, which contain helicase and RNA polymerase sequence motifs, together comprise the essential virus-encoded components of BSMV RNA replicase. A second BSMV protein (beta b) which contains a helicase motif is not required for RNA replication. A small cysteine-rich protein (gamma b) is dispensable for infection of plants, but in its absence the accumulation of viral coat (beta a) and beta b proteins is significantly reduced. In addition, mutations in both the gamma b and gamma a (replicase) proteins can affect the systemic movement phenotype.
Drug Combinations↗
Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts.
Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27 degrees but were barely detectable at 32 degrees. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.
Blotting, Northern↗
Replication of sonchus yellow net virus in infected protoplasts.
Tobacco (Nicotiana edwardsonii and N. benthamiana) protoplasts infected with the plant rhabdovirus sonchus yellow net virus (SYNV) were found to be suitable for studies of replication. SYNV messenger RNAs could be detected within 2 hr postinoculation (PI), accumulated to a maximum within 24 hr, and subsequently declined to undetectable levels by 60 hr. Plus- and minus-sense genomic RNAs appeared later and were most abundant by 36 hr PI, but the levels decreased by 60 hr. The four major SYNV structural proteins could be detected by Western blot serological analyses by 24 hr PI and were present in highest concentrations between 43 and 60 hr PI. Among various glycosylation inhibitors, only tunicamycin treatment of protoplasts altered viral protein patterns and resulted in synthesis of a glycoprotein with an apparent molecular weight (Mr) 10% smaller than that found in untreated protoplasts. Two specific cleavage products of the nucleocapsid protein estimated to be 21,000 and 37,000 Mr appeared in N. benthamiana-infected protoplasts by 60 hr PI, but in the presence of tunicamycin, the cleavage products were evident by 38 hr. This result suggests that specific cleavage of the N protein is correlated with stress of infected cells due to viral accumulation and/or tunicamycin treatment.
Blotting, Western↗
Induction by HMBA and DMSO of genes introduced into mouse erythroleukemia and other cell lines by transient transfection.
We have found rapid induction of various genes, including human globin genes, in response to hexamethylene bisacetamide (HMBA) and dimethyl sulfoxide (DMSO) in transiently transfected cells. In mouse erythroleukemia cells (MELCs), this effect is detected within 1 hr of exposure of the cells to inducer before the endogenous mouse globin genes are induced. It does not require protein synthesis and is reversed if the inducer is removed. This and other evidence suggest that the mechanism involves a change in activity of a factor intimately involved with transcription, probably as a result of post-translational modification. As such, it may represent an early triggering event in terminal differentiation, and its relevance to the expression of human globin genes in stable transfectants and to induction of the mouse globin genes is discussed. Other cell lines (K562 and NSO) also show this response, which may therefore involve a ubiquitous mechanism. We also found that HMBA depresses the expression of endogenous globin genes in K562, the opposite of this differentiation inducer's effect on MELC.
Acetamides↗
Dose response in growth of pigs injected daily with porcine somatotropin from 57 to 103 kilograms.
Exogenous administration of porcine somatotropin (PST) has been shown to promote growth in the pig; however, dose-response relationships and interactions with PST source and sex for animals taken to market weight have not been established clearly. The present study was conducted to determine the relationship between dosage of pituitary-derived and recombinantly manufactured PST (pPST and rPST) and growth (ADG), structural soundness, gain-to-feed ratio (G/F) and average daily feed intake (ADF). Crossbred barrows (n = 113) and gilts (n = 97) were injected with either saline, 1.5, 3.0, 6.0 or 9.0 mg/d of pPST or rPST from 57 +/- .3 to 103.5 +/- .7 kg live weight. Pigs were housed five per pen and had ad libitum access to an 18% crude protein diet for the duration of the experiment. Response curves for pPST and rPST did not differ (P greater than .20) for ADG, soundness score or ADF. Although regression coefficients for response of G/F to pPST and rPST differed (P = .05), pairwise comparisons of treatment means did not (P greater than .10). Response curves for barrows and gilts did not differ for ADG or soundness (P greater than .05). Averaged over PST source and sex, quadratic dose-responses were detected for ADG, G/F and ADF (P less than .01), but PST had no effect on soundness (P greater than .25). Exponential regression models best described the dose-response relationships, and 6.0 mg.pig-1.d-1 was predicted to result in 95% of maximal achievable response for days to 103.5 kg and G/F. At this dose, pigs were predicted to grow .15 kg/d (20%) faster during the treatment period, reach slaughter weight 11.6 d earlier, consume .56 kg/d (19%) less feed, and have a G/F .095 (36%) greater than controls.
Animals↗
Human papillomavirus in women with vulvar intraepithelial neoplasia III.
Untreated cases of vulvar intraepithelial neoplasia (VIN) III may progress to invasive vulvar carcinoma. Tissues from 29 New Zealand women with VIN III were examined for the presence of human papillomavirus (HPV) types 6, 11, 16 and 18 by in situ hybridization and polymerase chain reaction. HPV 16, the only HPV type detected in the lesions, was identified in about half the cases. HPV-positive women were younger than HPV-negative women, and their lesions displayed koilocytosis more often. In four of five cases in which there was a progression to invasive cancer, HPV 16 was detected in both the VIN III and invasive cancer tissue.
Age Factors↗