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At least 1,279 records · Page 71Linked to original sources
PubMed1997

Glycosyl-phosphatidylinositol-phospholipase type D: a possible candidate for the generation of second messengers.

Membrane associated glycosyl-phosphatidylinositols have been shown to be the precursors of inositol phosphoglycan second messengers. Extraction of human liver membranes and purification by serial thin layer chromatography revealed three glycolipids which co-migrated with glycosyl-phosphatidylinositol from rat liver. These lipidic fractions were partially sensitive to treatment with nitrous acid and to hydrolysis by glycosyl-phosphatidylinositol-specific phospholipase D from bovine serum. In parallel, glycosyl-phosphatidylinositol isolated from rat liver was found to be a substrate for the enzyme generating a biologically active inositol phosphoglycan species (determined by measuring inhibition of protein kinase A activity and stimulation of cell proliferation within the chicken embryo cochleovestibular ganglion). This molecule was recognised by an anti-inositol phosphoglycan antibody. Hence, we propose that glycosyl-phosphatidylinositol-specific phospholipase D could be implicated in cellular signalling.

Animals↗
PubMed1999

A homologous radioimmunoassay for the measurement of urotensin II in the euryhaline flounder, Platichthys flesus.

A sensitive and specific homologous radioimmunoassay (RIA) has been developed to measure tissue and circulating levels of the fish caudal neurosecretory system neuropeptide, urotensin II (UII), in the euryhaline flounder Platichthys flesus. A polyclonal antiserum was raised against flounder UII in rabbit; UII-125I was produced by the iodogen method and purified by HPLC. Antiserum specificity to flounder UII was demonstrated through lack of cross-reactivity with several small peptides and parallelism with standard curves for serial dilutions of UII in plasma and urophysial extracts. Biological activity of the peptide measured by UII RIA was confirmed by bioassay. Plasma intra- and interassay coefficients of variation were 9 and 18% (n = 5 and n = 3), respectively, nonspecific binding constituted 4.6% (+/-1.42%, n = 8) of total counts, and the limit of RIA detectability was estimated as 1.5 x 10(-16) M UII/assay tube. Plasma samples were subject to a reversed-phase liquid chromatography purification protocol which had an extraction efficiency of 63% (+/-10%, n = 6) and showed consistent recovery of UII over a range of plasma volumes and peptide concentrations. Plasma UII concentrations in seawater (SW)-adapted flounder (3.80 +/- 0.77 x 10(-11) M, n = 7) were significantly higher than those in freshwater (FW)-adapted fish (1.10 +/- 0.15 x 10(-11) M, n = 7). This variation coincided with differences in plasma osmolality and Na+ levels. No differences were found, however, between urophysial UII concentrations in SW-adapted (3.71 +/- 1.78 x 10(-10) M UII/gland, n = 7) and FW-adapted (2.53 +/- 1.33 x 10(-10) M UII/gland, n = 7) flounder.

Adaptation, Physiological↗
PubMed1980

Effect of iodoxamide ethyl on allergy skin tests.

Lodoxamide ethyl is a new cromolyn-like drug which prevents antigen-induced mediator release from mast cells and antigen-induced bronchoconstriction in sensitive animals and man. The purpose of this study was to determine the effect of a single administration of lodoxamide ethyl on allergy skin tests. The effect of this drug on allergy skin testing was studied in a double-blind design on ten adult subjects allergic to ragweed. Serial end-point titrations with short ragweed extract and with histamine were performed after a placebo and 1- and 3-mg capsules of lodoxamide ethyl. The immediate wheal-and-flare responses as well as the late allergic reaction were recorded. No statistically significant difference was found between lodoxamide ethyl and placebo in the suppression of the allergen-induced immediate wheal-and-flare response or the late allergic reaction. Furthermore, 1 and 3 mg of oral lodoxamide ethyl did not inhibit the histamine-induced cutaneous reactions.

Amino Acids↗
PubMed1995

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Animals↗
PubMed2005

Anaplasma platys: an improved PCR for its detection in dogs.

This study compares two PCR assays for the detection of Anaplasma platys in dog blood using primers based on the A. platys 16S rRNA gene. The first approach utilized a "standard" PCR protocol composed of a "single-step" direct amplification using an Ehrlichia genus-specific primer set. The second assay being a "nested" PCR screen that first involved a universal bacterial primer set that amplified the majority of the 16S rRNA gene, followed by the nested round of PCR using an A. platys-specific primer set. Of the 22 dogs sampled, 10 were found to contain A. platys DNA using both protocols, and an additional two dogs were found positive using the nested technique. An extract of A. platys positive genomic DNA was serially diluted and comparison of sensitivities determined between the nested PCR, and a direct assay using A. platys-specific primers. The nested protocol demonstrated an increased sensitivity by at least 2 orders of magnitude when compared to the direct assay alone. Our results indicated that the nested PCR assay with its increased sensitivity would be useful for experimental research investigations as well as offer the potential for use as a routine test in diagnostic pathology.

Anaplasma↗
PubMed2006

Analysis of gene expression profiles of normal human nasal mucosa and nasal polyp tissues by SAGE.

BACKGROUND: A systemic determination of gene expression profiles in nasal polyp compared with normal nasal mucosa would contribute considerably to the investigation of the disease marker in various rhinopathy and general knowledge on the formation of human nasal polyp. OBJECTIVE: The aim of this study was to identify the transcriptome of the normal human nasal mucosa and nasal polyp by the serial analysis of gene expression. METHODS: mRNA was extracted from normal inferior turbinate mucosa and nasal polyp. Short sequences (tags), each one usually corresponding to a distinct transcript, was isolated and concatemerized into long DNA molecules, which were cloned and sequenced. RESULTS: We detected 65,305 tags for normal nasal mucosa and 55,829 tags for nasal polyp, representing 20,629 and 17,636 potential transcripts species, respectively. Of the unique tags encountered more than once, 92% (normal nasal mucosa) and 90% (nasal polyp) matched known genes or expressed sequence tags, whereas the remainder did not match any GenBank sequences. Therefore, 504 and 539 novel transcripts were identified in normal nasal mucosa and nasal polyp, respectively. Although the expression levels of most transcripts in both libraries were similar, 114 transcripts were differentially expressed at a statistically significant level (P < .05)-that is, 65 and 49 transcripts among them were expressed at a higher level in normal nasal mucosa and nasal polyp, respectively. CONCLUSION: This information should be very useful for basic knowledge as well as for future studies on pathophysiological conditions of human nasal mucosa, providing some clues to evaluate the altered factors in a variety of rhinopathies. CLINICAL IMPLICATIONS: The results of this study might contribute to general knowledge on the formation of human nasal polyp.

Adult↗
PubMed2005

Annotating nonspecific SAGE tags with microarray data.

SAGE (serial analysis of gene expression) detects transcripts by extracting short tags from the transcripts. Because of the limited length, many SAGE tags are shared by transcripts from different genes. Relying on sequence information in the general gene expression database has limited power to solve this problem due to the highly heterogeneous nature of the deposited sequences. Considering that the complexity of gene expression at a single tissue level should be much simpler than that in the general expression database, we reasoned that by restricting gene expression to tissue level, the accuracy of gene annotation for the nonspecific SAGE tags should be significantly improved. To test the idea, we developed a tissue-specific SAGE annotation database based on microarray data (). This database contains microarray expression information represented as UniGene clusters for 73 normal human tissues and 18 cancer tissues and cell lines. The nonspecific SAGE tag is first matched to the database by the same tissue type used by both SAGE and microarray analysis; then the multiple UniGene clusters assigned to the nonspecific SAGE tag are searched in the database under the matched tissue type. The UniGene cluster presented solely or at higher expression levels in the database is annotated to represent the specific gene for the nonspecific SAGE tags. The accuracy of gene annotation by this database was largely confirmed by experimental data. Our study shows that microarray data provide a useful source for annotating the nonspecific SAGE tags.

Cell Line↗
PubMed2001

Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin.

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.

Animals↗
PubMed1955

An infectious hepatitis of undetermined origin in mice. I. Description of the disease.

Serial intraperitoneal passage in mice of a saline extract of the pooled livers, spleens, and kidneys of such animals has led to the demonstration after three or more passages of a transmissible agent causing hepatitis. The mice developed an illness after 3 to 4 weeks characterized by hepatosplenomegaly, and serous ascites. Spontaneous diuresis and recovery usually occurred during the subsequent 2 to 4 weeks. Histological studies of the livers showed diffuse mononuclear infiltrations, focal accumulations of mononuclear cells, perivascular mononuclear cuffing, dilated sinusoids, and occasionally focal areas of necrosis. Mice which have recovered from the disease showed no noteworthy resistance to it, and their sera failed to protect against the infectious agent. Attempts to infect rabbits, guinea pigs, monkeys, and embryonated hens' eggs yielded negative results, but young hamsters developed the disease in mild form.

Animals↗
PubMed1995

Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA.

Restriction endonuclease analysis of the DNA extracted orf virus strain NZ2, which had been serially passaged in primary bovine testis cells, revealed a population of variants that had over-grown the wild-type virus. At least three distinct mutant forms were identified in which the right end of the genome had been duplicated and translocated to the left end, accompanied by deletions of sequences at the left end. Sequencing of a single variant isolated from the heterogeneous population revealed that recombination had occurred between non-homologous sequences. In this case, 6.6 kb of DNA at the left end of the genome had been replaced by 19.3 kb from the right end. The transposition resulted in the deletion at the left end of 3.3 kb of DNA encoding three genes and the terminal sequences of a fourth gene. The three genes completely deleted were a homologue of dUTPase, a gene that encodes a protein containing ankyrin-like repeats and a homologue of the 5K gene of the vaccinia virus WR strain. Experimental inoculation of sheep showed that the genes are also non-essential in vivo, but that the size of the lesion was reduced, compared with that induced by the wild-type, and resolved more rapidly.

Animals↗
PubMed1994

Flavanones with potent antibacterial activity against methicillin-resistant Staphylococcus aureus.

With the therapeutic concept of using the defensive ability of plants against microbial infections, phytoalexin, an antimicrobial phytochemical was studied for its ability to inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Extracts from Sophora exigua (Leguminosae) were fractionated by serial chromatography and the anti-MRSA activity of each fraction was determined by the agar-plate method. Among the active isolates, 5,7,2',6'-tetrahydroxy-6-isoprenyl-8-lavandulyl-4'-methox yflavanone (exiguaflavanone D) completely inhibited the growth of all the MRSA strains examined at the concentration of 1.56-6.25 micrograms mL-1, and 5, 2',6'-trihydroxy-8-lavandulyl-7-methoxy-flavanone (exiguaflavanone B) inhibited at a concentration of 50 micrograms mL-1. This former compound is expected to be a phytotherapeutic agent for MRSA infections as an alternative to conventional antibiotics with unwanted side-effects or the appearance of antibiotic-resistant bacteria.

Anti-Bacterial Agents↗
PubMed1993

Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.

Animals↗
PubMed1995

Augmented urokinase receptor expression in atheroma.

Smooth muscle cell proliferation and migration into neointima are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated. One potential mediator of both smooth muscle cell proliferation and migration is activation of plasminogen by activators bound to receptors on cells within the vessel wall. To determine whether vascular smooth muscle cells within atheroma express the receptor for urokinase-type plasminogen activator (uPA-R), we characterized atheroma in cholesterol-fed New Zealand White rabbits and human subjects by immunostaining. Intense immunostaining of uPA-R was observed throughout the neointima in both rabbit and human atherosclerotic lesions with the use of a monoclonal antibody to uPA-R. uPA-R was not detectable in normal arterial tissues. uPA-R was localized to macrophages and neointimal smooth muscle cells identified by immunostaining in serial sections. Furthermore, uPA-R protein in extracts from atheroma was present in at least a ninefold greater quantity compared with extracts from normal vessels, as shown by Western blotting. Expression of uPA-R mRNA in migrating vascular smooth muscle cells did not increase significantly. Thus, altered posttranscriptional regulation may be contributing to the increased uPA-R. In vitro, antibodies to uPA-R delayed the migration of cultured vascular smooth muscle cells. Our results suggest that increased cell-surface uPA-R contributes to pericellular proteolysis and consequently increased neointimalization secondary to increased vascular smooth muscle cell migration in atheroma.

Animals↗
PubMed1991

Airway blood flow modifies allergic airway smooth muscle contraction.

We tested the hypothesis that airway perfusion modifies the contractile response of airway smooth muscle to allergen challenge by influencing the clearance of locally released spasmogens. In six intact, lightly sedated, sheep allergic to Ascaris suum, we measured tracheal mucosal blood flow (Qtr) with a soluble gas uptake method and tracheal dead space (Vtr), an index of airway smooth muscle tone, by helium dilution before and serially after local aerosol challenge with A. suum extract or ragweed extract (control). The former challenge was repeated during continuous intravenous infusion of either vasopressin or nitroglycerin, which by themselves had no effect on Vtr and decreased and increased Qtr, respectively. Ragweed had no effect on Qtr and Vtr, whereas A. suum increased mean (+/- SE) Qtr by 111 +/- 31% (p less than 0.05) and decreased mean Vtr by 15 +/- 2% (p less than 0.05) immediately after challenge, with Qtr returning to baseline by 40 min and Vtr by 80 min. Vasopressin infusion prevented the A. suum-induced increase in Qtr and prolonged the decrease in mean Vtr (p less than 0.05). During nitroglycerin infusion, A. suum failed to alter Qtr or Vtr. Vasopressin and nitroglycerin had no effect on the contractile responses of tracheal smooth muscle to A. suum in vitro. These results indicate that the effects of vasopressin and nitroglycerin on antigen-induced airway smooth muscle contraction in vivo were due to alterations in airway blood flow rather than to alterations in the release of or airway smooth muscle responsiveness to chemical mediators.

Acetylcholine↗
PubMed1975

Extraction of collagenase from the 6000 times g sediment of uterine and skin tissues of mice. A comparative study.

An enzyme capable of digesting native collagen in solution at neutral pH was extracted from the 6 000 times g sediment of the involuting uterus of the mouse and of the back skins of mice and rats. The collagenase could be dissociated at cold-room temperature from the sediment in about equal amounts when neutral Tris buffer containing 1.0M NaCl or 5M urea was used for the extraction step. The enzyme has been concentrated by ammonium sulfate precipitation and the activity was measured by using [14C]collagen in solution at pH 7.5. Collagen breakdown products were identified by disc electrophoresis. The amount of enzyme extracted was a function of temperature and salt concentration. As 5M urea extracted collagenase from the sediment in a relatively short time, this method of extraction seems to be a useful tool for serial experiments in the study of collagenase activity in collagen-rich tissues.

Aging↗
PubMed1988

Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squamous cell carcinomas of the cervix.

The sensitivity of human papilloma virus type 16 (HPV-16) DNA detection by DNA in situ hybridization using biotinylated probes (bio-DISH) was estimated by performing this technique on snap-frozen tissue sections of 10 cervical squamous cell carcinomas containing increasing amounts of HPV-16 as determined by Southern blot hybridization. A protocol using serial sections for bio-DISH and DNA extraction was used. The number of positively stained cells and the detection limit were strongly dependent on the treatment of the sections with proteinase K prior to hybridization. At low proteinase K concentration (0.1 micrograms/ml), the detection limit appeared to be 30-40 HPV-16 DNA copies per carcinoma cell, whereas morphology was preserved. A high proteinase K concentration (1-5 micrograms/ml) often resulted in an increase in the number of positively stained cells but also in a poor morphology. The detection limit was improved to at least 20 HPV-16 DNA copies per carcinoma cell.

Biotin↗
PubMed1989

A new method of transcervical female sterilization: preliminary results in rabbits.

In search of an outpatient procedure for female sterilization, the authors performed the following animal study. Two groups of seven rabbits were subjected to laparotomy, hysterotomy, and hysteroscopy. The uterotubal junction (UTJ) was destroyed with bipolar electrical current, and a plug (Aqualloy, Drachten, The Netherlands) was inserted. The plug on the right side contained quinacrine (Q) in group A and platelet extract (PE) in group B. Histologic assessment by serial sections indicated occlusion of the UTJ in all but one case (96% success rate). This one failure is attributed to the technical difficulties associated with hysteroscopy in the rabbit. The authors project that this method may have the potential of becoming an acceptable outpatient procedure for female sterilization in the human.

Animals↗
PubMed1980

N-isopropyl-[123I] p-iodoamphetamine: single-pass brain uptake and washout; binding to brain synaptosomes; and localization in dog and monkey brain.

The kinetics of N-isopropyl-p-[123I]iodoamphetamine in rat brains were determined by serial measurements of brain uptake index (BUI) after intracarotid injection; also studied were its effects on amine uptake and release in rat's brain cortical synaptosomes; and its in vivo distribution in the dog and monkey. Serial BUI correspond to a first-pass extraction efficiency of 100% and a washout half-time of approximately 318 sec. The S-(+)-isomer inhibited norepinephrine uptake in synaptosomes as strongly as D-amphetamine, but was more potent in inhibiting synaptosomal uptake of serotonin. Both isomers were comparable to D-amphetamine in causing release of serotonin from synaptosomes, but the S-(+)-isomer promoted release of dopamine, whereas D-amphetamine did not. No specific localization in brain nuclei of the dog was seen, but there was progressive accumulation in the eyes. Rapid initial brain uptake in the ketamine-sedated monkey was noted, and further slow brain uptake occurred during the next 20 min but without retinal localization. High levels of brain activity were maintained for several hours. The quantitative initial single-pass clearance of the agent in the brain suggests its use in evaluation of regional brain perfusion. Its interaction with brain amine-binding sites suggests its possible application in studies of cerebral amine metabolism.

Amphetamines↗