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Attenuation of allergen sensitivity early in the course of ragweed immunotherapy.

During the course of an open immunotherapy (IT) study of ragweed (RW)-allergic patients, nasal mediator release was studied by provocation testing. All subjects had a history of seasonal RW rhinitis, positive skin puncture test to RW, and RW-specific IgE by RAST. Nasal challenge was performed with serial dilutions of RW extract, before and after 12 weekly injections, providing a cumulative dose of 0.22 microgram of Amb a I. Serum IgE and IgG and basophil histamine release with RW were also measured. By 12 weeks of IT, when only 1% of the usual maintenance level dose had been administered, mean histamine release and TAME-esterase activity in nasal washes decreased significantly (p less than 0.05 and p less than 0.01). Prostaglandin D2 release did not change. Skin sensitivity decreased (p less than 0.05), whereas RW-specific IgE increased (p less than 0.05). No significant change in basophil histamine release was observed for RW or a control antigen. Only six of 40 subjects had an RW-specific IgG rise greater than 0.05 microgram/ml. Changes in nasal sensitivity did not correlate with the increases in IgE or IgG or with the change in skin test sensitivity. These present data indicate that there is a significant decline in nasal sensitivity to inhaled RW very early in the course of IT. There is, however, no indication of a relationship between the decreased nasal sensitivity and the production of RW-specific IgG antibodies.

Adolescent↗

Radioimmunoassays for fish tail neuropeptides: I. Development of assay and measurement of immunoreactive urotensin I in Catostomus commersoni brain, pituitary, and plasma.

Antisera were raised in rabbits using highly purified urotensin I (UI) or UI (4-41) conjugated with high-molecular-weight proteins. Antiserum 2U4, at a final dilution of 1:300,000, gave 50% binding to iodinated UI (specific activity 40-120 microCi/micrograms). Carp (Cyprinus carpio) and sucker (Catostomus commersoni) UI peptides cross-reacted completely with the antiserum. Results from cross-reactivity tests using various tryptic and Staphylococcus aureus protease fragments of UI indicated that the antigenic sites of the main antibody(ies) are directed against a part sequence in the C-terminal region of the peptide although antibodies with lower concentration or affinity, recognizing N-terminal region(s) of the peptide, are also present. No cross-reactivity was seen with insulin, secretin, vasoactive intestinal peptide or a structural UI-homolog, the ovine hypothalamic corticotropin-releasing factor, up to molar concentrations. Sauvagine, another structurally homologous peptide from frog skin, showed only 0.1% cross-reactivity. The urophysis-specific proteins, urophysins B and D, showed a low degree of cross-reactivity. Assays of serial dilutions of urophysis extracts from teleostean species yielded parallel displacement curves, except in the case of the pacific goby, Gillichthys mirabilis. Varying concentrations of immunoreactive UI were present in different regions of C. commersoni brain, spinal cord, and pituitary, and also in plasma. The measurement of 17.5 +/- 2.1 fmol UI/ml of plasma indicates that this assay may be used for the study of circulating UI peptide(s).

Amino Acid Sequence↗

Radioimmunoassay of methionine(5)-enkephalin sulphoxide: phylogenetic and anatomical distribution.

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine(5)-enkephalin (Met(5)-Enk), Met(5)-Enk sulphoxide (Met(5)-Enk-S), has been developed. Antisera were raised in rabbits against Met(5)-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (125I) Met(5)-Enk bound to antiserum by Met(5)-Enk was poor, but Met(5)-Enk-S displayed good displacement suggesting that the Met(5)-Enk immunogen was oxidised to Met(5)-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met(5)-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine(5)-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met(5)-Enk (2--5) sulphoxide and between 9% and 25% with D-Ala(2)-Met(5)-Enk sulphoxide. The antisera showed less than 0.01% cross-reactivity with other Met(5)-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met(5)-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met(5)-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met(5)-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met(5)-Enk, and may account for the relative insensitivity of some published RIAs using Met(5)-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met(5)-Enk IMR.

Animals↗

Characterization of alpha-melanocyte-stimulating hormone in rat pancreas.

Reverse-phase high performance liquid chromatography and radioimmunoassay were used to characterize alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in rat pancreas. Relative to synthetic alpha-MSH standards, serial dilutions of pancreas extracts showed parallel and concentration dependent displacement of (125I) alpha-MSH from alpha-MSH antibody. Chromatographic separation revealed immunoreactive material coeluting with synthetic N,O-diacetyl alpha-MSH, which accounted for 78% of total alpha-MSH materials in this tissue. The remainder of immunoreactive alpha-MSH coeluted with synthetic alpha-MSH, desacetyl alpha-MSH, or their methionine sulfoxides. In contrast with anterior pituitary, it appears that biosynthetic processing of alpha-MSH from pro-opiomelanocortin (POMC) may be similar in rat pancreas and pituitary intermediate lobe, since their relative alpha-MSH immunoreactive elution profiles were similar. These findings support the hypothesis of tissue specific regulation of biosynthetic processing of POMC.

Animals↗

Dendroaspis natriuretic peptide and its functions in pig ovarian granulosa cells.

Dendroaspis natriuretic peptide (DNP), a 38-amino acid peptide, was isolated from the venom of green mamba. It has structural and functional similarities to the other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP is present in pig ovarian granulosa cells and to define its biological functions. The serial dilution curves of extracts of granulosa cells and follicular fluid were parallel to the standard curve of DNP, and a major peak of molecular profile of both extracts by HPLC was synthetic DNP. The concentration of DNP was 7.51+/-1.46 pg/10(7) cells and 24.81+/-2.38 pg/ml in granulosa cells and follicular fluid, respectively. Natriuretic peptides increased cGMP production in the purified membrane of granulosa cells with a rank order of potency of C-type natriuretic peptide (CNP)>atrial natriuretic peptide (ANP)=DNP. mRNAs for natriuretic peptide receptor-A (NPR-A), NPR-B and NPR-C were detected by RT-PCR. The binding site of (125)I-DNP was also observed in granulosa cell layer by in vitro autoradiography. Synthetic DNP inhibited the secretion of ANP from granulosa cells in a concentration-dependent manner and the potency was similar to CNP. The concentration of DNP and CNP, which inhibited the secretion of ANP by 50%, was about 1 nM. Increases in production of cGMP in granulosa cells were observed by DNP or CNP. Therefore, these results show the existence of DNP system and the cross-talk between natriuretic peptides in pig ovarian granulosa cells.

Animals↗

Dendroaspis natriuretic peptide system and its paracrine function in rat colon.

Dendroaspis natriuretic peptide (DNP), a 38-amino-acid peptide, was isolated from the venom of Green Mamba. It has structural and functional similarities to other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP system is present in the rat colon and to define its biological functions. The serial dilution curve of extracts of colonic tissues was parallel to the standard curve of DNP and a major peak of molecular profile by HPLC was synthetic DNP. The concentration of DNP was 0.5 +/- 0.04 ng/g of colonic tissues. DNP as well as atrial natriuretic peptide and C-type natriuretic peptide caused dose-dependent increases in cGMP production in the purified membrane of colonic tissues. Three types of natriuretic peptide receptor mRNAs were detected using semi-quantitative RT-PCR. Functionally, synthetic DNP inhibited the spontaneous contraction of rat colonic circular muscle in a concentration-dependent manner. The potency appeared to be at least 10 times greater than that of CNP. Furthermore, DNP inhibited carbachol-induced muscle contraction, suggesting that it also can modulate the nerve regulation of colonic motility. This study demonstrates the presence of DNP system in rat colon and its function as a local regulator of colonic motility.

Animals↗

Distribution and volumetric assessment of initial approximal caries lesions in human premolars and permanent molars using computer-aided three-dimensional reconstruction.

Serial sections from 21 extracted premolars and permanent molars, divided into three age groups (group 1, 10-19 years; group 2, 20-39 years; group 3, 50-69 years.), were viewed by polarized light microscopy for reconstruction of the caries lesions. The volumes of the lesional body and the transparent zone, and the volumes of early dentinal lesions were calculated; and an enamel demineralization index (EDI) and an enamel-dentine demineralization index (EDDI), representing the volumetric ratio of the body of the lesion to the translucent zone, and the body of the lesion to the volume of the early dentinal lesion, respectively, were determined. The method showed that there are three typical sites of individual initial caries lesions at the approximal surface: within the contact area, in the subcontact area, and in the cervical area. Volumetric assessment demonstrated a larger volume of the body of the lesion in older teeth with a smaller volume of the translucent zone relative to the lesional body. From these results it can be concluded that there are usually more than one individual initial caries lesions at the approximal tooth surface. Calculation of demineralization indices demonstrated different features for small lesions with pronounced transparent zones and for large lesions with small transparent zones. The computer-assisted three-dimensional reconstruction technique and the volumetric assessment are of value in investigations of the micromorphology and progression of natural caries lesions in permanent teeth.

Adolescent↗

Application of touchdown enzyme time release (TETR)-PCR for diagnosis of Chlamydophila abortus infection.

Chlamydophila abortus-DNA was detected using a touchdown enzyme time-release (TETR)-polymerase chain reaction (PCR) assay as an improved test for sensitive and rapid diagnosis of abortion in small ruminants. Two hundred and fifty two placentae, liver or spleen tissue samples from aborting ewes and goats or aborted lambs and kids in which C. abortus infection was suspected were examined by TETR-PCR and the results were compared with cell culture. Sixty-five tissue samples were found to be TETR-PCR positive while only 56 samples were cell culture-positive. After resolution of discrepant samples with a confirmatory nested PCR assay, TETR-PCR had a sensitivity of 97% and a specificity of 99.5% while culture had a sensitivity of 84.8% and a specificity of 100%. The analytical sensitivity of the TETR-PCR assay was determined with DNA extracted from 4-fold serial dilution of C. abortus B577 culture and found to be 0.25 inclusion-forming unit per PCR. No reduction in the analytical sensitivity was noted when the assay was tested with mouse liver samples spiked with 4-fold serial dilution of C. abortus B577 culture. No target product was amplified when DNA from Chlamydophila pecorum was tested. TETR-PCR used in this study is a practical, rapid, sensitive and specific assay that could be used for the detection of C. abortus in infected tissue samples. We recommend the use of this assay as a supplemental diagnostic tool for detection of C. abortus in infected tissue samples.

Abortion, Veterinary↗

Skin sensitivity to allergen does not accurately predict airway response to allergen.

BACKGROUND: Allergen challenge of the asthmatic airway has become widely applied in the study of allergic asthma in humans. Skin sensitivity correlates with inhaled sensitivity in some populations. Skin test titration has been proposed as a useful tool to guide the selection of initial allergen concentration. OBJECTIVE: To determine the relationship between skin test sensitivity and inhaled reactivity to allergen. METHODS: We examined the relationship between skin test and inhaled reactivity in 22 allergic asthmatic subjects. Methacholine bronchoprovocation was performed using a standardized tidal breathing technique. Prick skin test titrations were done using serially diluted lyophilized antigen extracts reconstituted in normal saline from 1:100,000 to 1:10. Inhaled allergen challenge was routinely performed in the morning using the same allergen employed in skin test titration. RESULTS: There was no correlation between skin test threshold and the inhaled concentration required to produce a 20% fall in FEV1 (r = 0.07; P = .78). If subjects who manifested marked cutaneous reactivity (i.e., skin reactivity at dilutions greater than 1:10,000) were excluded from analysis, there was a significant correlation between cutaneous and inhaled reactivity (r = 0.84; P < .001). CONCLUSION: While a correlation between skin test threshold and inhaled reactivity is present in some subjects with allergic asthma, the relationship is inconsistent.

Administration, Inhalation↗

Essentiality of ubiquinone for choline oxidation in rat liver mitochondria.

Rat liver mitochondria treated extensively with n-pentane are incapable of oxidizing choline. Choline oxidation is more sensitive than is succinate oxidation to serial n-pentane extraction of mitochondria. The ability to oxidize choline is restored by the addition of ubiquinone-2 or ubiquinone-10 to the oxidase assay medium.

Alkanes↗

Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16.

E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity.

Animals↗

Antibodies to an Epstein-Barr virus nuclear antigen synthetic peptide in infectious mononucleosis. Report of two cases.

The Epstein-Barr virus nuclear antigen (EBNA-1) contains a region of repeating glycine and alanine amino acids. It has been shown that this region contains a major epitope of EBNA-1. With well-characterized sequential sera from two cases of acute infectious mononucleosis, a specific IgM response was detected to the EBNA-1 synthetic peptide by enzyme-linked immunosorbent assay (ELISA). Conversely, an IgG response was observed in the convalescent phase of the illness with a progressive decline of the IgM antibodies. This response was observed with heterophil-positive and heterophil-negative EBV/IM. The peptide-specific serologic response was confirmed by immunoblotting, the serial serum samples on extracts of EBV transformed B-cells. There was excellent correlation between the antipeptide ELISA and blotting techniques.

Acute-Phase Reaction↗

Stratified communities of active Archaea in deep marine subsurface sediments.

Archaeal 16S rRNA was extracted from samples of deep marine subsurface sediments from Peru Margin site 1227, Ocean Drilling Program leg 201. The amounts of archaeal 16S rRNA in each extract were quantified by serial dilution and reverse transcription (RT)-PCR. The results indicated a 1,000-fold variation in rRNA content with depth in the sediment, with the highest concentrations found near the sediment surface and in the sulfate-methane transition zone (SMTZ). The phylogenetic composition of the active archaeal population revealed by cloning and sequencing of RT-PCR products changed with depth. Several phylotypes affiliated with marine benthic group B (MBGB) dominated clone libraries from the upper part of the SMTZ and were detected only in this layer. Members of the miscellaneous crenarchaeotal group (MCG) dominated clone libraries from the other layers. These results demonstrate that archaeal communities change in activity and community composition over short distances in geochemically distinct zones of deep subseafloor sediments and that these changes are traceable in the rRNA pool. It was shown for the first time that members of both the MCG and MBGB Archaea are more active in the SMTZ than in layers above and below. This indicates that they benefit either directly or indirectly from the anaerobic oxidation of methane. They also appear to be ecophysiologically flexible, as they have been retrieved from a wide range of marine sediments of various geochemical properties.

Archaea↗

In vivo diagnosis of fissure caries using a new electrical resistance monitor.

The aim of this investigation was to study the accuracy of a newly developed electrical resistance monitor in diagnosing occlusal fissure caries in vivo in teeth without cavitation. The electrical resistance was measured in vivo on human third molars without existing restorations and without any macroscopic carious cavitation. Data from 41 sites on 26 occlusal surfaces were obtained. After measurement, the teeth were extracted, histologically prepared, and serially sectioned perpendicular to the occlusal surface and examined for the presence of caries. Nineteen sites had no caries, 7 had enamel caries, and 15 sites showed dentinal caries, 4 of which deep dentinal caries. For the diagnosis of occlusal caries with the electrical resistance monitor, the specificity was 0.77, the sensitivity 0.93, and the accuracy 0.83. Likelihood ratios for four diagnostic levels (no caries, enamel caries, dentinal caries, and deep dentinal caries) were 0.09, 0, 3.47, and 4.16, respectively. Diagnosis of occlusal caries using conventional bite-wing radiographs showed a specificity of 0.77, a sensitivity of 0.62, and an accuracy of 0.71. The electrical resistance monitor was well suited to detect in vivo occlusal caries under clinically intact fissures. The rather high value (0.23) of false-positive ratings, however, might lead to a substantial number of sound teeth being restored unnecessarily. Substantial improvement of occlusal, caries diagnosis may be achieved by combining this method with others.

Adult↗

Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines.

OBJECTIVE: To determine relative detection rates and detection limits for 6 published polymerase chain reaction (PCR) assays used for detection of feline herpesvirus type 1 (FHV-1) DNA. SAMPLE POPULATION: 5 vaccines licensed for use in preventing FHV-1-associated disease; 15 conjunctival biopsy specimens collected from cats with keratitis, conjunctivitis, or both; and a plaque-purified field isolate of FHV-1 cultured in vitro. PROCEDURE: Vaccines and clinical samples were assessed for FHV-1 DNA by use of all 6 assays. Detection rates were calculated by assuming that any sample in which FHV-1 DNA was detected was a true-positive result. Detection limits were estimated by use of serial dilutions of DNA extracted from cultured FHV-1 and 1 clinical sample. RESULTS: Testing by use of all 6 assays resulted in detection of FHV-1 DNA in all 5 vaccines. Testing by use of all 6 assays yielded concordant results for 9 of 15 conjunctival biopsy specimens (8 with negative results and 1 with a positive result). Calculated detection rates for clinical samples ranged from 29% to 86%. Assay sensitivity was ranked similarly by use of detection rate or detection limit. CONCLUSIONS AND CLINICAL RELEVANCE: Testing by use of all assays was equally likely to detect vaccine virus. Therefore, a positive PCR result in a cat may reflect vaccine virus rather than wild-type virus. Test sensitivity as assessed by detection limits and detection rates varied greatly. Because FHV-1 can be shed in clinically normal animals, high detection rate will not necessarily correlate with high diagnostic sensitivity.

Animals↗

Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR.

The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture.

Animals↗

[Sectional anatomy and computer-assisted three-dimensional reconstruction of lateral skull base in normal].

OBJECTIVE: To investigate the sectional anatomy of lateral skull base in normal and provide data for diagnosis and selecting surgical approach of lesions of lateral skull base. METHOD: Three sets of coronal sections with plastination technique was made and investigated for the location, direction and mutual relationship in different section. Signals of some structures were extracted to reconstructed three-dimensional image. RESULT: The section with plastination displayed distinct nerve, muscle, vessel, bone in lateral skull base. The structures of lateral skull base were described in five typical section: the maxillary sinus, pharyngeal orifice of pharyngotympanic duct, foramen ovale, tympanie orifice of pharyngotympanic duet, inner auditory meatus. The three-dimensional images of sphenoid sinus, ethmoid sinus, trigeminal nerve, pharyngotympanic duet and inner carotid artery after extracted the signals in serial sections. CONCLUSION: Sectional anatomy with plastination contributed to understanding complicated anatomic structure including the location and mutual relationship, three dimensional image can vividly display the structure underlying lateral skull base surgery.

Humans↗

A review of the literature on the etiology of Hodgkin's disease.

Extensive research to trace the cause of Hodgkin's disease to a bacterial or protozoan agent has proven fruitless. Although a viral cause for Hodgkin's disease has been previously suggested, early explorations along that line have not been confirmed. With the development of newer techniques for the study of viral characteristics certain apparently significant factors in Hodgkin's disease have been encountered. Most promising has been the consistent demonstration that Seitz-filtered, sterile Hodgkin's disease lymph node extract can be passed serially in fertile chicken eggs and that the amniotic fluid from these eggs possesses the capacity to interfere with the growth of influenza virus in eggs.

Hodgkin Disease↗