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PubMed1983

European elk papillomavirus: characterization of the genome, induction of tumors in animals, and transformation in vitro.

The European elk papillomavirus (EEPV) genome was cloned in the BamHI cleavage site of the pBR322 vector. The cloned genome was used for construction of a physical map, employing restriction endonucleases BamHI, BglII, HindIII, PvuII, SacI, and XhoI. The sequence homology between the EEPV and bovine papillomavirus type 1 genomes was elucidated by performing hybridizations in different concentrations of formamide. Sequence homology could only be revealed under less stringent conditions, i.e., Tm - 43 degrees C. Nucleotide sequence information was also collected from the regions which lie adjacent to the three HindIII sites that are present in the EEPV genome. The results made it possible to align the EEPV and bovine papillomavirus type 1 genomes. Transformation by EEPV was demonstrated with the C127 mouse cell line, and fibrosarcomas were induced in young hamsters after subcutaneous injection. The transformed cells and the tumors contain multiple, nonintegrated copies of the EEPV genome. Virus particles could not be detected either in tumors or in transformed cells.

Animals↗
PubMed1984

Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands.

Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. We reexamined the uniqueness of minus strand encapsidation for the autonomous parvoviruses. Although we found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNA when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.

Animals↗
PubMed1981

In vitro translation of the intermediate filament proteins desmin and vimentin.

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.

Animals↗
PubMed1988

Expression of the X-CGD gene during induced differentiation of myeloid leukemia cell line HL-60.

The expression of the X-CGD gene, which encodes the heavy-chain subunit of the phagocyte cytochrome b, was studied during induced myeloid differentiation of HL-60 cells. Incubation of the cells with a combined regimen of retinoic acid and dimethyl formamide resulted in granulocytic morphological differentiation and acquisition of nitroblue tetrazolium reduction, a measure of superoxide generation. During the 5-day course of induced differentiation, the levels of X-CGD mRNA transcripts rose 13-fold, with a 2-fold increase detectable within 3 h of exposure to retinoic acid. Relative transcription rates for the X-CGD gene, determined by nuclear runoff, increased two- to eightfold after 24 to 72 h of induced differentiation. However, the greater change in X-CGD mRNA levels than that in transcription rates implies the involvement of posttranscriptional regulation as well. Fractionation by centrifugal elutriation into phases of the cell cycle showed expression of X-CGD transcripts predominantly in G1 cells before induction and in all phases of the cell cycle 24 h after induction. Thus the rapid increase in X-CGD expression in induced cells reflects the acquisition of functional competence and not the concomitant cessation of proliferation or shift in cell cycle distribution.

Cell Differentiation↗
PubMed1988

Non-isotopic detection of in situ nucleic acid in cervix: an updated protocol.

Recently, sensitive non-isotopic in situ hybridisation (NISH) methodology for the detection of human DNA and human papilloma virus (HPV) DNA in archival paraffin blocks of cervix was described. An amended protocol, now used in this laboratory for detection of these genes by NISH is presented. The amendments include the following: protease digestion at 37 degrees C; tissue dehydration in air rather than ethanol; stringency washing in formamide solution; blocking non-specific binding of avidin alkaline phosphatase with a modified buffer; and increasing the concentration of avidin alkaline phosphatase for detecting low abundance DNA. These changes simplify and increase the sensitivity of the protocol such that "Y" chromosome repeats are visualised in almost all female cells.

Cervix Uteri↗
PubMed1993

Selective digestion of mouse chromosomes with restriction endonucleases. Oligonucleotide priming of single-stranded DNA produced with exonuclease III.

L-929 mouse chromosomes prepared for electron microscopy have been treated with MspI, EcoRI, and HaeIII restriction endonucleases (REs). RE-induced nicks were amplified with exonuclease III to obtain single-stranded DNA (ss-DNA) motifs. The ss-DNA produced was enough to permit hybridization of a series of random oligonucleotides. These can be used as primers, which are extended by the Klenow fragment using non-isotopic labelled dUTP. The incorporation of biotinylated dUTP is detected with a gold-tagged streptoavidin as the reporter molecule. This allows, in mouse chromosomes, the detection of different rates of sensitivity to the digestion with specific REs in distinct intraheterochromatic DNA subsets. In addition, these results show that enzymatic production of ss-DNA seems to be adequate for electron microscopy work since the chromatin fiber is preserved better than in denatured DNA produced with heat, NaOH, or formamide.

Animals↗
PubMed1979

Analysis of a 5S RNA-protein complex isolated from the ribosomes of rye embryos.

Rye embryo ribosomes were dissociated into subunits and the large subunit fraction was treated with formamide. A low molecular weight complex of RNA and protein (RNP) was released. Electrophoresis of the RNP in polyacrylamide gels containing sodium dodecyl sulphate yielded an RNA band and a single protein band. The protein had a molecular weight of approximately 41 000 and the RNA of the complex was shown to be 5S ribosomal RNA. Embryos were germinated in the presence of [32P]orthophosphate and the labelled RNP was isolated from their ribosomes. The RNA component was partially digested with pancreatic A ribonuclease and the parts protected from degradation by the protein were determined by sequence analysis. Although the whole 5S RNA molecule was shielded to some extent, the portion most protected was between nucleotides 68 and 108. This is, therefore, probably the part of plant cytosol 5S RNA which is primarily involved in the interaction with protein in the complex and possibly in the ribosome as well.

Base Sequence↗
PubMed1982

Injection defect in alkylated and depurinated T7 bacteriophage: analysis by DNA ejection.

Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid-DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

Alkylation↗
PubMed1986

Human platelet osmotic water and nonelectrolyte transport.

The osmotic water (Pf) and nonelectrolyte permeability (Ps) properties of human platelets were characterized using the stopped-flow light-scattering technique. At 37 degrees C, Pf = 0.007 +/- 0.001 cm/s, the urea reflection coefficient (sigma urea) = 0.95 +/- 0.04, and Ps for a series of permeant nonelectrolytes was (in cm X s-1 X 10(-6)) 2.1 (urea), 3.5 (glycerol), 3.8 (thiourea), 17 (ethylene glycol), 18 (acetamide), 23 (formamide), and 24 (butyramide). Pf did not depend on the size of the osmotic gradient or on the direction of volume flow. Mercurial sulfhydryl reagents did not inhibit osmotic water transport, and phloretin and phenylurea did not inhibit urea transport. There was a discontinuity in the temperature dependence for both Pf and urea permeability (P urea) at 36 degrees C; enthalpy (delta H) = 25 (greater than 36 degrees C) and 4.4 kcal/mol (less than 36 degrees C) for Pf, and delta H = 26 (greater than 36 degrees C) and 7 kcal/mol (less than 36 degrees C) for P urea. In contrast to the facilitated water and urea transport systems in the red blood cell, these results suggest that the mechanism for water and urea transport in the platelet is primarily by diffusion through membrane phospholipid. A computer-simulated model of platelet circulation through the renal medulla, based on the measured values for Pf, P urea, and sigma urea, indicated that platelets undergo an approximately 40% decrease in volume in the inner medulla and an approximately 20% overshoot in volume as they return to the external isosmotic environment.

Amides↗
PubMed1993

Urea alters set point volume for K-Cl cotransport, Na-H exchange, and Ca-Na exchange in dog red blood cells.

Urea equilibrates rapidly across the red blood cells of mammals. It was speculated that urea might affect the cell volume sensor by virtue of its properties as a protein perturbant. At concentrations of 0.1-0.6 M, urea caused a decrease in the set points for shrinkage-induced Na-H exchange, swelling-induced K-Cl cotransport, and swelling-induced Ca-Na exchange of dog red blood cells. Okadaic acid opposed the action of urea on all three pathways. The effects were reversible and not due to cyanate. Formamide and acetamide had actions similar to urea but not as potent. Equimolar concentrations of methanol had no effect. The coordinated influence of urea on three separate volume-activated transporters suggests that it acts on a mutual regulatory system that senses and transduces volume stimuli.

Amides↗
PubMed1996

Apical membrane permeability of MDCK cells.

The osmotic water permeability (Pf) and permeability to nonelectrolytes were determined for the apical membrane of clonal strain Madin-Darby canine kidney (MDCK) C12 cells cultured as cysts with the apical membrane facing the surrounding medium. Pf and solute permeabilities were calculated from the rate of volume change of cysts by digitizing images at 1-s intervals after instantaneous osmotic challenge. Image measurement was fully automated with the use of a program that separated the image of the cyst from the background by using adaptive intensity thresholding and shape analysis. Pf, calculated by curve fitting to the volume loss data, averaged 2.4 +/- 0.1 micron/s and was increased by addition of amphotericin B. The energy of activation for Pf was high (16.3 kcal/mol), and forskolin (50 microM) had no effect on Pf. Two populations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The Pf of multicell cysts was the same as single cell cysts, suggesting that paracellular water flow is not significant. Solute permeability was measured using paired osmotic challenges (sucrose and test solute) on the same cyst. Urea permeability was not different from zero, whereas the permeabilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epithelial apical membrane. The combination of the unique model of MDCK apical-out cysts and fully automated data analysis enabled determination of apical membrane permeability in intact epithelial cells with high precision.

Amphotericin B↗
PubMed2002

Expression of TASK-1, a pH-sensitive twin-pore domain K(+) channel, in rat myocytes.

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K(+) channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.

Animals↗
PubMed2006

Characterization of blood pressure and renal function in chromosome 5 congenic strains of Dahl S rats.

The present study examined whether transfer of overlapping regions of chromosome 5 that include (4A+) or exclude the cytochrome P-450 (CYP) 4A genes from the Lewis rat alters the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and/or the development of hypertension in congenic strains of Dahl salt-sensitive (S) rats. The expression of CYP4A protein and the production of 20-HETE in the renal outer medulla was greater in the 4A+ congenic strain than the levels seen in S rats or in overlapping control congenic strains that exclude the CYP4A region. Mean arterial pressure (MAP) rose from 122 +/- 2 to 190 +/- 7 mmHg in S rats and from 119 +/- 2 and 123 +/- 2 to 189 +/- 7 and 187 +/- 3 mmHg in the two control congenic strains fed an 8.0% NaCl diet for 3 wk. In contrast, MAP only increased from 112 +/- 2 to 150 +/- 5 mmHg in the 4A+ congenic strain. Chronic blockade of the formation of 20-HETE with N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide (TS-011; 1 mg/kg bid) restored the salt-sensitive phenotype in the 4A+ congenic strain and MAP rose to 181 +/- 6 mmHg after an 8.0% NaCl dietary challenge. TS-011 had no effect on the development of hypertension in S rats or the two control congenic strains. The pressure-natriuretic and diuretic responses were fivefold greater in the 4A+ congenic strain than in S rats. These results indicate that transfer of the region of chromosome 5 between markers D5Rat108 to D5Rat31 from the Lewis rat into the Dahl S genetic background increases the renal production of 20-HETE, improves pressure-natriuresis and opposes the development of salt-induced hypertension.

Animals↗
PubMed1986

Water permeability and fluidity of renal basolateral membranes.

Water and nonelectrolyte permeability in basolateral membrane vesicles (BLMV) isolated from rat and rabbit renal cortex were characterized. Osmotic water (Pf) and solute (Ps) permeabilities were determined from the time course of scattered light intensity in response to osmotic gradients. In rabbit BLMV, Pf = 2.5 X 10(-2) cm/s, Purea = 1.2 X 10(-6) cm/s, and sigma urea = 0.95 (23 degrees C). Relative solute permeabilities were urea, 1; thiourea, 0.83; ethylene glycol, 9.7; glycerol, 4.6; formamide, 20; and acetamide, 12. Pf and Purea were not altered by organic mercurials, phloretin, urea, and high-affinity urea analogues. delta H (12-50 degrees C) was 10.7 and 2.5 kcal/mol for Purea and Pf, respectively; in contrast, reported delta H for Pf in rabbit brush-border membrane vesicles (BBMV) was 2 kcal/mol (T less than 33 degrees C) and 14 kcal/mol (T greater than 33 degrees C). To examine whether membrane fluidity changes were associated with this difference, fluorescence anisotropy decay was measured using 1,6-diphenyl-1,3,5-hexatriene (DPH). In both BLMV and BBMV, DPH rotation was hindered at all temperatures (5-53 degrees C). Changes in steady-state anisotropy were attributable to changes in DPH rotational freedom rather than to changes in DPH rotational rate without evidence for a definitive membrane thermotropic phase transition. These results suggest that BLMV urea transport occurs by lipid diffusion and that osmotic water transport is rapid and may be facilitated. A comparison of transepithelial Pf with BLMV and BBMV Pf is consistent with transcellular osmotic water flow in the proximal tubule.

Animals↗
PubMed1988

Renal metabolic/excretory coupling.

Renal metabolic/excretory coupling is the enhancement of urinary excretion dependent upon renal metabolism. The nitrofurothiazoles, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), are model compounds used to study metabolic/excretory coupling. FANFT is deformylated to ANFT by renal deformylase enhancing ANFT excretion. In the rat, ANFT excretion after oral FANFT administration was 100-fold greater than ANFT excretion when ANFT was administered. FANFT and ANFT uptake into purified proximal tubules achieved equilibrium within 60 s and was demonstrated in nonviable tubules. FANFT partitioned into oil better than ANFT. Albumin inhibited FANFT and ANFT uptake into oil and decreased tubular uptake by 65%. Tubular FANFT uptake was threefold or greater than that of ANFT uptake with or without albumin. Renal deformylase was predominantly cytosolic and yielded apparent Km and Vmax of 6.7 microM and 6.1 nmol ANFT.min-1.mg protein-1, respectively. Deformylase activity was abolished by boiling, was specific for N-formylated compound, and was not altered by dinitrophenol treatment. Renal metabolic/excretory coupling for FANFT/ANFT combines energy-independent uptake with metabolism (deformylation), resulting in enhanced urinary ANFT excretion.

Animals↗
PubMed1993

Neuropeptide Y inhibits neurogenic inflammation in guinea pig airways.

We examined the effect of neuropeptide Y (NPY) on neurogenic airway microvascular leakage. Male Dunkin-Hartley guinea pigs (250-350 g) were anesthetized with urethan (2 g/kg ip). The cervical artery and vein were cannulated for monitoring blood pressure and injecting drugs, respectively. Atropine and propranolol (each 1 mg/kg i.v.) were administered 30 min before the experiment. After pretreatment with saline (vehicle for NPY) or NPY (1-100 micrograms/kg i.v.), Evans blue dye (30 mg/kg iv) was administered. Then, bilateral vagal nerves were electrically stimulated (5 V, 7 Hz, 5-ms duration for 3 min) to induce airway plasma leakage. Airways were divided into four sections [trachea (Tr), main bronchi, central intrapulmonary airways (IPA), and peripheral IPA] and incubated in formamide (37 degrees C for 16 h). The concentration of Evans blue dye was measured by spectrophotometer. Furthermore, we examined the effect of NPY on exogenous substance P- (0.3 microgram/kg i.v.) induced plasma extravasation. Bilateral vagal stimulation significantly increased leakage of dye in Tr to peripheral IPA. NPY did not affect basal leakage but did significantly inhibit neurogenic plasma extravasation in a dose-dependent manner with maximal inhibitions of 42.3 (Tr), 67.7 (main bronchi), 38.2 (central IPA), and 26.3% (peripheral IPA) at 30 micrograms/kg. Exogenous substance P-induced plasma extravasation was not inhibited by NPY. We conclude that NPY inhibits neurogenic inflammation by prejunctional inhibition of neuropeptide release from airway sensory nerve terminals.

Adrenergic beta-Antagonists↗
PubMed2006

Slow diffusion of K+ in the T tubules of rat cardiomyocytes.

Cardiomyocyte contractility is regulated by the extracellular K(+) concentration ([K(+)](o)). Potassium dynamics in the T tubules during the excitation-contraction cycle depends on the diffusion rate of K(+), but this rate is not known. Detubulation of rat cardiomyocytes was induced by osmotic shock using formamide, which separated the surface membrane from the T tubules. Changes in current and membrane potential in voltage-clamped (-80 mV) and current-clamped control and detubulated cardiomyocytes were compared during rapid switches between 5.4 and 8.1 mM [K(+)](o), and the results were simulated in a mathematical model. In the voltage-clamp experiments, the current changed significantly slower in control than in detubulated cardiomyocytes during the switch from 5.4 to 8.1 mM [K(+)](o), as indicated by the times to achieve 25, 50, 90, and 95% of the new steady-state current [control (ms) t(25) = 98 +/- 12, t(50) = 206 +/- 20, t(90) = 570 +/- 72, t(95) = 666 +/- 92; detubulated t(25) = 61 +/- 11, t(50) = 142 +/- 17, t(90) = 352 +/- 52, t(95) = 420 +/- 69]. These time points were not significantly different either during the 8.1 to 5.4 mM [K(+)](o) switch or in current-clamped cardiomyocytes switching from 5.4 to 8.1 mM [K(+)](o). Mathematical simulation of the difference current between control and detubulated cardiomyocytes gave a t-tubular diffusion rate for K(+) of approximately 85 mum(2)/s. We conclude that the diffusion of K(+) in the T tubules is so slow that they constitute a functional compartment. This might play a key role in local regulation of the action potential, and thus in the regulation of cardiomyocyte contractility.

Action Potentials↗
PubMedDate not supplied

Mutation analysis of TP53 exons 5-8 by automated constant denaturant capillary electrophoresis.

DNA fragments melt characteristically according to their nucleotide sequence and length, when exposed to denaturants such as temperature, urea or formamide. Small differences within a defined sequence, like a base mutation, will result in a slightly different melting behavior of the aberrant DNA fragment compared to that of the wild type sequence. This feature has previously been exploited for mutation detection by constant denaturant capillary electrophoresis (CDCE). In this report, we describe an automated approach (ACDCE) using a commercially available apparatus (ABI 310 Genetic Analyzer) to analyze mutations in exons 5-8 of TP53. The running conditions were determined by temperature titration of the fragments on the apparatus, and an operating sensitivity showed that 0.1% mutated alleles could be detected against a background of wild-type alleles. Up to 48 samples can be analyzed by ACDCE without any need for operator intervention. The apparatus is commercially available, and there is no need for instrument modification. To our knowledge this is the first report on the analysis of TP53 exons 5-8 by ACDCE.

Automation↗