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At least 1,045 records · Page 58Linked to original sources
PubMed1992

A quantitative solid-phase enzymeimmunoassay for 13,14-dihydro-15-keto- prostaglandin F2 alpha in plasma.

Enzymeimmunoassays (EIA) can be viable alternatives to radioimmunoassays (RIA). Indeed, from an environmental perspective, EIA are preferable to RIA. Therefore, the purpose of this project was to develop a quantitative EIA for 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in bovine plasma. Acetylcholine esterase bound covalently to PGFM, rabbit anti-PGFM, mouse monoclonal anti-rabbit IgG, and PGFM were the principle reagents used for the EIA. Validation experiments indicated that: 1) PGFM standard curves, with doses ranging from 391 to 200,000 fg per microtiter well, were linear; 2) assay sensitivity averaged 391 fg per well; 3) for satisfactory results, PGFM had to be extracted from plasma; 4) content of PGFM in ethyl ether extracts of aliquots from serial dilutions of whole plasma with unknown amounts of PGFM and charcoal-stripped plasma supplemented with known amounts of PGFM did not deviate from parallelism with PGFM standard curves in buffer; 5) correlation between EIA and RIA measurements of PGFM in the same plasma samples was .95; 6) the regression of EIA data on RIA data was linear (Y = .93 x + 83.9; r2 = .91); 7) intra- and interassay coefficients of variation were 3.3 and 10.6%, respectively. The EIA developed in this project is a valid and reliable method for quantitating PGFM in extracts of bovine plasma.

Animals↗
PubMed1984

Myosin changes in hypertrophied human atrial and ventricular myocardium. A correlated immunofluorescence and quantitative immunochemical study on serial cryosections.

Two antigenically distinct types of myosin heavy chain, referred to as alpha and beta, have been identified in autoptic and bioptic specimens of human heart using specific antimyosin antibodies. By immunofluorescence heavy chain alpha was present in all atrial myocytes and in a variable number of ventricular myocytes. Heavy chain beta was present in all ventricular myocytes and in a number of atrial myocytes. Ventricular hypertrophy in patients with aortic stenosis, systemic hypertension or tetralogy of Fallot was characterized by an almost complete absence of fibres reactive with anti-alpha. A striking decrease in alpha chain reactivity and a parallel increase in beta chain reactivity was apparent in the hypertrophied left atria of patients with mitral stenosis. To quantify these myosin changes a novel procedure was developed whereby myosin was extracted from single cryosections serial to those processed for immunofluorescence and the relative amount of alpha and beta heavy chain was determined by enzyme immunoassay. Heavy chain alpha was less than 5% in most normal ventricular specimens and disappeared completely under the effect of pressure overload. On the other hand heavy chain beta was generally undetectable in the left atrial myocardium but increased up to 90% in biopsies of hypertrophied atria.

Adult↗
PubMed2006

Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen.

To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.

Animals↗
PubMed2002

Experimental study on a novel compound extracted from Traditional Chinese Medicine for treatment of alveolar echinococcosis.

OBJECTIVE: To evaluate the efficacy of a novel compound derived from Traditional Chinese Medicine (TCM) for treatment of alveolar echinococcosis in the Kuming mouse. METHODS: The compound was extracted from TCM by serial procedures including amalgamation of TCM drugs, boiling in a beaker, cold condensation using a reflux instrument, evaporation by mild heating and finally, formation of a deep brown powder. An animal model of alveolar echinococcosis was established concurrently. A bit of alveococcus tissue derived from alveolar echinococcosis in the mouse was transplanted into the peritoneal cavity of the musculus of a Kuming mouse. The duration of infection after inoculation was classified as either 1 week or 10 weeks; the animals were again divided into treated groups and control groups. Estimation of the alveococcus inhibitory rate and electron microscopic observation served as the main methods for assessment of efficacy. The infected mice in the treated groups were intra-gastrically administered once per day with the TCM extractive powder at doses of 20 mg.kg(-1) for three months continuously. All the animals were sacrificed and examined half a month after the end of TCM treatment. RESULTS: Total wet weight of alveococcus collected from the 9 mice in the treated group after one week of infection was 37.8 g (x +/- s, 4.196 +/- 2.090 g), which was markedly lower than the amount collected from 10 mice of the control group (121.3 g, x +/- s, 12.1 +/- 4.3 g). Therefore, the inhibitory rate of the alveococcus was 65.7% (P < 0.01). Similarly, the total wet weight of alveococcus from 7 mice of the treated group after 10 weeks of infection was 4.3 g (x +/- s, 0.62 +/- 1.21 g), which was much lower than the amount collected from 6 mice of the control group (17.85 g, x +/- s, 2.93 +/- 3.28 g). The inhibitory rate of alveococcus was 80.6% (P < 0.01). Furthermore, the ultrastructural appearances of alveococcus showed obvious differences between the two groups. CONCLUSION: TCM extractive powder for treatment of murine alveococcosis is considered to be a hopeful anti-echinococcus compound; it is a topic for further study.

Animals↗
PubMed1986

Vasopressin and oxytocin in the developing rat brain as shown by isoelectric focusing of radioimmunoassayable peptides.

Vasopressin and oxytocin content were measured by radioimmunoassay in tissue extracts from rat pituitary, hypothalamus and the extrahypothalamic brain from gestational day 14 until postnatal day 14. Assayable compounds were subsequently characterized by isoelectric focusing in gels. Fetal day 14 extracts contained only low amounts of both hormones (around 1-2 pg per fetus). Vasopressin content rapidly increased to levels of 41 ng in pituitary, 20 ng in hypothalamus, 0.3 ng in extrahypothalamic brain on postnatal day 14. Oxytocin content showed only measurable amounts around day 20 prenatally, reaching levels of, respectively, 10, 0.5 and 0.2 ng on postnatal day 14. The presence of radioimmunoassayable vasopressin in the extrahypothalamic brain on fetal day 14 (around 2 pg) confirms the early ontogeny of vasopressinergic innervation of the brain. Whenever measurable, immunoreactivity had an isoelectric point similar to that of synthetic vasopressin or oxytocin. The possibility that the detected amounts of vasopressin were due to vasotocin, which had been claimed to be present in fetal brain, was excluded by comparison of the regression coefficients of serial dilutions of the extracts in the radioimmunoassay with those of synthetic vasotocin or vasopressin.

Animals↗
PubMed1986

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts.

Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupal Manduca sexta (Lepidoptera) and larval Sarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (ED50) was 0.20 for Manduca and 0.15 for Sarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, when M. sexta larval prothoracic glands were challenged by S. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.

Animals↗
PubMed1994

Activity of extracts of Kigelia pinnata against melanoma and renal carcinoma cell lines.

Serial dilutions of standardised water, ethanol, and dichloromethane extracts of the stembark and fruits of Kigelia pinnata were tested for their growth inhibitory effects against four melanoma cell lines and a renal cell carcinoma line (Caki-2) using two different (MTT and SRB) assays. Lapachol, a possible constituent of these extracts, together with known therapeutic antineoplastic agents, was also tested in the same way. The IC50 of each extract was measured after extracts were diluted to 100 micrograms/ml in 1% ethanol or water. Significant inhibitory activity was shown by the dichloromethane extract of the stembark and lapachol (continuous exposure). Moreover, activity was dose-dependent, the extract being less active after 1 h exposure. Chemosensitivity of the melanoma cell lines to the stembark was greater than that seen for the renal adenocarcinoma line. In marked contrast, sensitivity to lapachol was similar amongst the five cell lines. Lapachol was not detected in the stembark extract.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Nucleophosmin/B23 regulates the susceptibility of human leukemia HL-60 cells to sodium butyrate-induced apoptosis and inhibition of telomerase activity.

Stable clones of HL-60 cells in which nucleophosmin/B23 was over-expressed or down-regulated were established. The nucleophosmin/B23 protein levels in nucleophosmin/B23 over-expressed (pCR3-B23) or down-regulated (pCR3-32B) cells during BuONa/vanadate-induced apoptosis were characterized as compared with control vector-transfected (pCR3) cells. Over-expression of nucleophosmin/B23 resulted in decreased susceptibility of the cells to BuONa/vanadate-induced apoptosis. The response to inhibition of telomerase activity under BuONa/vanadate treatment also decreased in nucleophosmin/B23 over-expressed (pCR3-B23) cells. On the other hand, down-regulation of nucleophosmin/B23 made the cells more susceptible to BuONa-induced apoptosis or inhibition of telomerase activity. More precisely, by serial dilutions of each extract, the telomerase activity of the cells without drug treatment was determined and was found to be higher in nucleophosmin/B23 over-expressed (pCR3-B23) cells and lower in nucleophosmin/B23 down-regulated (pCR3-32B) cells as compared with the control vector-transfected (pCR3) cells. Our results indicate that nucleophosmin/B23 plays a functional role in the control of cellular apoptosis and immortalization.

Apoptosis↗
PubMed2004

Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea.

Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.

Canada↗
PubMed1996

Persistence of vesicular stomatitis virus New Jersey RNA in convalescent hamsters.

Persistence of vesicular stomatitis virus New Jersey (VSV-NJ) was studied in experimentally infected hamsters (Mesocricetus auratus). We used reverse transcription and nested polymerase chain reaction (RT-NPCR) to probe tissues of hamsters inoculated with VSV-NJ Hazelhurst. Viral genomic RNA was detected in the brain, cerebellum, spleen, liver, kidney, and lung 2 months after infection, but only in the central nervous system at 10 and 12 months. Viral messenger RNA was detected in the brain of one hamster at 2 months after infection. Replicative intermediate was detected in the spinal cord of one hamster at 12 months. These results suggest that VSV-RNA persists in animals for long periods following infection, disease, and convalescence. However, infectious virus was not recovered from tissues by conventional serial passages of tissue extracts in Vero cells or by cocultivation.

Animals↗
PubMed1975

Radioimmunological determination of human C-peptide in serum.

A routing radioimmunoassay for human C-peptide in serum is described. Antibodies against human C-peptide were raised by immunizing guinea pigs with human b-component. Nine out of 12 animals produced useful antibodies within 6 months. Insulin antibodies coupled to Sepharose were used to bind human proinsulin and insulin in the serum and after centrifugation C-peptide was determined in the supernatant. The detection limit of the assay (calculated as 2 SD from zero) was about 0.003 pmole of C-peptide (in 100 mul). The main sources of error were: (1) Normal and diabetic sera devoid of C-peptide gave a displacement of 125I-Tyr-C-peptide varying from 0 to 0.16 nM (6 different antisera). Only one antiserum (M 1181) showed no displacement, and the values of C-peptide determined with this antiserum in normal and diabetic sera were lower than the values determined with another antiserum, which gave a value of 0.07 nM in the sera free of C-peptide. It is suggested that displacement found with most antisera is due to substances in serum that are not related to C-peptide or proinsulin. (2) Serial dilutions of pancreatic extracts and sera may yield dilution curves slightly different to those of the synthetic standard. Possible explanations are discussed. These sources of error can be eliminated or reduced by the proper selection of antisera. Fasting sera from 15 normals, 8 maturity-onset diabetics and 10 insulin-requiring diabetics showed the following concentrations of C-peptide: (M 1181) 0.35 +/- 0.09, 0.74 +/- 0.51 and 0.21 +/- 0.14 (nM, mean +/- SD). One hour after 1.75 g/kg oral glucose the values increased to 2.24 +/- 0.71, 2.34 +/- 0.24 nM.

Animals↗
PubMed1985

Hypercalcemia associated with increased circulating 1,25 dihydroxyvitamin D in a patient with pulmonary tuberculosis.

Studies are described in a 53-year-old man with far-advanced pulmonary tuberculosis who developed transient increases in circulating 1,25 dihydroxyvitamin D (1,25(OH)2D) and hypercalcemia while on antituberculous treatment. Serial dilution of an extract of the patient's serum obtained while he was hypercalcemic displaced [3H]-1,25(OH)2D3 from chick intestinal receptor in a manner identical to authentic 1,25(OH)2D3. Serum 25-hydroxyvitamin D (25OHD) was suppressed during the abnormal elevation of serum 1,25(OH)2D. It is concluded that tuberculosis is another chronic granulomatous disease in which hypercalcemia may result from abnormal metabolism of vitamin D.

Animals↗
PubMed2006

PLXDC1 (TEM7) is identified in a genome-wide expression screen of glioblastoma endothelium.

Glioblastomas are a highly aggressive brain tumor, with one of the highest rates of new blood vessel formation. In this study we used a combined experimental and bioinformatics strategy to determine which genes were highly expressed and specific for glioblastoma endothelial cells (GBM-ECs), compared to gene expression in normal tissue and endothelium. Starting from fresh glioblastomas, several rounds of negative and positive selection were used to isolate GBM-ECs and extract total RNA. Using Serial Analysis of Gene Expression (SAGE), 116,259 transcript tags (35,833 unique tags) were sequenced. From this expression analysis, we found 87 tags that were not expressed in normal brain. Further subtraction of normal endothelium, bone marrow, white blood cell and other normal tissue transcripts resulted in just three gene transcripts, ANAPC10, PLXDC1(TEM7), and CYP27B1, that are highly specific to GBM-ECs. Immunohistochemistry with an antibody for PLXDC1 showed protein expression in GBM microvasculature, but not in the normal brain endothelium tested. Our results suggest that this study succeeded in identifying GBM-EC specific genes. The entire gene expression profile for the GBM-ECs and other tissues used in this study are available at SAGE Genie (http://cgap.nci.nih.gov/SAGE). Functionally, the protein products of the three tags most specific to GBM-ECs have been implicated in processes critical to endothelial cell proliferation and differentiation, and are potential targets for anti-angiogenesis based therapy.

Brain Neoplasms↗
PubMed1996

Racial variations in the extent of tooth root translucency in ageing individuals.

One hundred and ninety-eight single-rooted teeth from individuals aged 21-90 yr of Caucasian, Malay, Chinese Malay and Indian Malay origin were studied. Single or serial longitudinal sections of extracted teeth were cut following dye imbibition of patent dentinal tubules. The extent of sclerosis of apical dentinal tubules was assessed and correlated with the age of the individual. Correlation with age was higher in the Caucasian than the Malay races and within the Malaysian racial groups correlation with age was highest in the Malays and lowest in the Chinese. It is concluded that factors other than age may be important in the formation of sclerotic apical dentine in teeth of different racial origin. The effect of racial origin should be considered when using sclerosis as a means of age determination in forensic cases.

Adult↗
PubMed1987

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells.

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.

Ataxia Telangiectasia↗
PubMed1992

Human thymocytes secrete luteinizing hormone: an autocrine regulator of T-cell proliferation.

There is evidence that there may be bidirectional communication between immune and gonadotropin producing cells. In the present study we describe the secretion and function of an LH like peptide secreted from the human thymus. Serial dilutions of thymic extracts obtained from surgically removed thymic tissues displaced I125 LH from LH antiserum parallel to that produced by pituitary LH. Thymic LH was detected as early as 10 days of age and maximal concentrations were noted at 5 months. Utilizing a sensitive and specific hormonal enzyme linked immunoplaque assay developed in our laboratory, we were able to detect LH secretion from unstimulated human thymocytes. Neutralizing endogenously released LH in PHA stimulated thymocytes with LH antiserum significantly inhibited thymidine incorporation. We conclude that an LH-like peptide is secreted by human thymocytes which acts as an autocrine co-mitogen in lymphoproliferation.

Cell Division↗
PubMed1984

Partial characterization of immunoreactive substance P in the rat pituitary gland.

Two distinct carboxy-terminus-directed anti-substance P (SP) sera (R-1C and R-6G) were used to characterize immunoreactive SP (I-SP) in acetic acid extracts of anterior pituitary (AP) and posterior pituitary (PP) glands of adult male rats. The tissue concentrations of I-SP measured by R-1C and R-6G were comparable. The contents of I-SP were 600-1150 pg/AP and 25-52 pg/PP. I-luteinizing hormone releasing hormone and I-somatostatin (I-SOM) were undetectable in AP extracts, but PP extracts contained the equivalents of 325-785 pg I-SOM/gland. Serial dilutions of AP and PP extracts produced displacement curves with both SP antisera that were parallel to the respective synthetic SP standard and hypothalamic extract displacement curves. Gel filtrations of AP and PP extracts on a Sephadex G-25 column produced I-SP peaks eluting in the same fractions as synthetic SP and hypothalamic I-SP. However, the AP I-SP profile also revealed a side peak migrating between the void volume and the major I-SP peak. Neither immunoreactive species in the AP extract were eliminated when eluted with 6.0 M guanidine HCl, a strong denaturing agent. In vitro incubation of paired anterior hemipituitaries for 30 min in the presence of a 56 mM K+ concentration resulted in a significant (p less than .0001), 25-fold increase in the release of I-SP into the incubation medium above the mean control value. Radiofrequency lesions placed in the median eminence-arcuate region of male rats caused a significant (p less than .001) reduction of I-SP in both the AP and PP. These reductions were inversely related to the plasma prolactin values. The elevation in plasma prolactin was taken as an index of completeness of lesions. We conclude that: 1) the rat pituitary contains I-SP as assessed by its immunologic and chromatographic behavior, 2) K+ depolarization is a potent stimulator of the release of AP I-SP in vitro, 3) the ME-arcuate region is important for the maintenance of pituitary I-SP levels in the rat.

Amino Acid Sequence↗
PubMed1980

Protective effect opf lodoxamide tromethamine on allergen inhalation challenge.

Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell-mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with lodoxamide (0.01, 0.1, or 1.0 mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers significant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and 1.0 mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen. Side effects observed at these doses were minimal.

Adolescent↗