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PubMed1985

Serum neutralization of cytotoxin from Pasteurella haemolytica, serotype 1 and resistance to experimental bovine pneumonic pasteurellosis.

Sera from several groups of experimental calves were tested for cytotoxin neutralizing capacity. The relationship between this capability and resistance of the animals to an experimental challenge was examined. All undiluted bovine sera tested, other than fetal bovine serum, neutralized cytotoxin. Preadsorption of selected sera with formalin-killed P. haemolytica did not reduce their neutralizing capacity. Crude IgG fractions extracted from bovine sera retained neutralizing capacity as well. Cytotoxin neutralization titers were determined by serial dilution of sera from cattle which were previously unexposed, naturally exposed, or exposed by vaccination to the organism. Both live and killed vaccines were used. Prior exposure to live organisms resulted in the production of antibodies to both cell surface antigens and cytotoxin, whereas exposure to the killed vaccine resulted in the production of antibodies primarily to cell surface antigens. Resistance to experimental challenge with the organism correlated directly with serum cytotoxin neutralizing titers.

Animals↗
PubMed1995

Identification of parent benzodiazepines by gas chromotography/mass spectroscopy (GC/MS) from urinary extracts treated with B-glucuronidase.

Urinary glucuronide metabolites of the benzodiazepines were converted back to the parent molecules after treatment with B-glucuronidase. The benzodiazepines were extracted by a one-step liquid/liquid extraction from urine or by a liquid/solid phase extraction. For the limit of detection (LOD), a standard solution of diazepam and oxazepam was serially diluted and analyzed to the point at which a reproducible analytical result was no longer obtained. Using a temperature program and a splitless mode of injection, excellent quantitation was achieved within an 8-min run time. Based upon specimens obtained from patients under a physician's care, we have determined that urinary concentrations of the benzodiazepines > 200 ng/ml are most likely due to abuse rather than to a prescribed ingestion under strict medical surveillance. Therefore, the calibration standard and cutoff concentration for a positive result was set at 200 ng/ml.

Alprazolam↗
PubMed1991

Culture of human outer root sheath cells from plucked hair follicles in serum-free conditions.

We succeeded in culturing human outer root sheath cells (ORSC) in serum-free conditions without a biological feeder layer. The combination of collagen type IV substrate and modified MCDB 153 medium supplemented with bovine pituitary gland extract (K-GM medium) could support the growth of ORSC. These cells can be serially cultivated for at least 4 passages and stored in liquid nitrogen with good recovery. Thus, a large series of experiments using ORSC may be run simultaneously.

Cell Division↗
PubMed2006

Purification of a novel aminopeptidase from the pollen of Parietaria judaica that alters epithelial integrity and degrades neuropeptides.

BACKGROUND: Parietaria judaica pollen is a common cause of pollinosis in the Mediterranean area. OBJECTIVE: This study sought to purify and characterize the peptidase responsible for the majority of proteolytic activity present in the pollen extract of P judaica, and to investigate its contribution to the allergic response. METHODS: A serial of chromatographic steps was applied to isolate the peptidase from P judaica's pollen, and its biochemical properties were determined. Bioactive peptides present in the airways were incubated with the peptidase, and their degradation was visualized by direct protein sequencing. In addition, we measured the cellular detachment, by methylene blue binding assay, of an airway-derived epithelial cell line (A549) in the presence of the peptidase, and visualized, by Western blot, the degradation of proteins from intercellular junctions. RESULTS: We purified a 98-kDa peptidase from the pollen of P judaica that was classified as an aminopeptidase on the basis of its biochemical properties and internal amino acid sequence. The aminopeptidase was able to degrade bioactive peptides. Moreover, the aminopeptidase caused cellular detachment of A549 cell line and degradation of occludin and E-cadherin. CONCLUSION: Our results suggest that the P judaica aminopeptidase can alter the integrity of the epithelium barrier by degrading occludin as well as E-cadherin. In addition, P judaica aminopeptidase can degrade bioactive peptides, which can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs. CLINICAL IMPLICATIONS: The novel aminopeptidase described here could constitute a relevant therapeutic target in the treatment of allergic disorders induced by the pollen of P judaica.

Amino Acid Sequence↗
PubMed2005

Echinacea alkamide disposition and pharmacokinetics in humans after tablet ingestion.

Echinacea is a widely used herbal remedy for the treatment of colds and other infections. However, almost nothing is known about the disposition and pharmacokinetics of any of its components, particularly the alkamides and caffeic acid conjugates which are thought to be the active phytochemicals. In this investigation, we have examined serial plasma samples from 9 healthy volunteers who ingested echinacea tablets manufactured from ethanolic liquid extracts of Echinacea angustifolia and Echinacea purpurea immediately after a standard high fat breakfast. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkamides were rapidly absorbed and were measurable in plasma 20 min after tablet ingestion and remained detectable for up to 12 h. Concentration-time curves for 2,4-diene and 2-ene alkamides were determined. The maximal concentrations for the sum of alkamides in human plasma were reached within 2.3 h post ingestion and averaged 336+/-131 ng eq/mL plasma. No obvious differences were observed in the pharmacokinetics of individual or total alkamides in 2 additional fasted subjects who took the same dose of the echinacea preparation. This single dose study provides evidence that alkamides are orally available and that their pharmacokinetics are in agreement with the one dose three times daily regimen already recommended for echinacea.

Adult↗
PubMed1991

Inhibition of pulmonary surfactant function by meconium.

The pathophysiology of meconium aspiration is marked by lung hyperinflation because of airway obstruction, which is often followed by an acute pneumonitis with classic lung injury characteristics. Surfactant dysfunction may contribute to this latter pulmonary pathophysiology. We sought to determine to what extent meconium itself might contribute to a functional surfactant deficiency. Specimens of newborn infants' first meconium were collected and pooled. Serial dilutions of the meconium were then added to various concentrations of calf lung surfactant extract, a mixture with the surface properties of natural surfactant that is used clinically to treat neonatal respiratory distress syndrome, and the dynamic surface activity of these mixtures was studied with a pulsating bubble surfactometer. At surfactant concentrations of less than or equal to 1.5 mg/ml, even 6500-fold dilutions of meconium-inhibited surface tension lowering ability (10 +/- 2 mN/m vs 1 +/- 0.1 mN/m for controls, p less than 0.05). Moreover, this inhibitory activity resided in both the chloroform-soluble and the aqueous phases of meconium and appeared to be additive in nature. However, at sufficiently high concentrations of surfactant, even large amounts of meconium were unable to affect surface tension lowering properties. Thus meconium inhibits surfactant function in a manner that is dependent on the surfactant concentration, suggesting the possible utility of exogenous surfactant therapy in some cases of meconium aspiration.

Animals↗
PubMed2001

Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts.

The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5 alpha-dihydrotestosterone (DHT) have anabolic effects on connective tissue and bone matrices. Human oral periosteal fibroblasts (HPF) and gingival fibroblasts (HGF) instigate healing in inflammatory periodontal lesions. The aim of this investigation was to compare the modulatory effects of I on the metabolism of two androgen substrates in human oral periosteal and gingival fibroblasts in culture. Monolayer cultures of both cell types (5(th)-9(th) passage) were established in Eagle's MEM and incubated with 14C-testosterone/14C-4-androstenedione and serial concentrations of I (0.5-50 microg/ml) for 24 h. The steroid metabolites were solvent extracted from the medium, separated by TLC and quantified using a radioisotope scanner. Both androgen substrates were metabolized mainly to DHT and 4-androstenedione/testosterone respectively, expressing 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity in both HPF and HGF. There were 51% and 73% increases in the levels of DHT over controls, with HGF and HPF respectively (n = 6; n = 4, P < 0.01) in response to I at 1-5 microg/ml, often reaching control values at 50 microg/ml. The expression of 17 beta-HSD activity showed less stimulation than the levels of DHT. Both androgen substrates were effective in this metabolic conversion, which is applicable to healing responses in both males and females in vivo. There were 57% increases (n = 4; P < 0.01) over controls, in the formation of androstanediol from 14C-4-androstenedione at 10 microg of I, in HPF. This transformation may regulate androgen action in androgen-dependent tissue. In addition to its anti-inflammatory properties, indomethacin can contribute to anabolic reparatory responses, by increasing the expression of steroid metabolizing enzymes in gingival and periosteal fibroblasts, in the inflammatory periodontal lesion.

Adult↗
PubMed1992

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.

Animals↗
PubMed1989

Endogenous Gibberellins and Shoot Growth and Development in Brassica napus.

Greenhouse-grown oilseed rape (Brassica napus, annual Canola variety ;Westar') plants were harvested at six dates from the vegetative phase until the early pod (silique)-fill/late flowering stage. Endogenous gibberellin (GA)-like substances were extracted from stems, purified, and chromatographed on silica gel partition columns prior to bioassay in serial dilution using the ;Tan-ginbozu' dwarf rice microdrop assay. The concentrations of total endogenous GA-like substances were low during vegetative stages (1 nanogram GA(3) equivalents/gram dry weight), and rose 300-fold by the time of floral initiation. After floral initiation the concentration of GA-like substances fell, then rose again during bolting to maximal levels during the early pod-fill stage (940 nanograms per gram dry weight). The qualitative profiles of GA-like substances varied across harvests, with higher proportions of a GA(1)-like substance at the early pod-fill stage. In a second study stems were similarly harvested at eight dates and the concentrations of endogenous GA(1), the principal bioactive native GA of oilseed rape, were determined by gas chromatography-selected ion monitoring using [17,17-(2)H]GA(1) as a quantitative internal standard. The concentration of GA(1) increased at about the time of floral initiation and then subsequently fell, thus confirming the pattern noted above for total GA-like substances. The exogenous application of paclobutrazol (PP333), a persistent triazole plant growth regulator (PGR) which blocks GA biosynthesis, or another triazole, triapenthenol (RSW0411), prevented flowering as well as bolting; plants remained at the vegetative rosette stage. These results imply a causal role for endogenous GA, in the control of bolting, which normally precedes anthesis. Further, the rise in the concentration of total endogenous GA-like substances, including GA(1), which was associated with floral initiation, and the prevention of visable floral development by the triazole PGRs, also indicates a role for endogenous GAs in the regulation of flowering in B. napus.

Journal Article↗
PubMed1998

Preliminary report: laboratory-induced stain removal as assessed by environmental scanning electron microscopy.

Environmental scanning electron microscopy (ESEM) was employed to observe stain removal during brushing with Arm & Hammer Dental Care and Crest Regular Toothpaste. ESEM allows serial examinations of the same sample, and does not require a destructive preparative process. Three extracted molars were cleaned, placed into a 96-hour broth culture of Streptococcus mutans, and stain was produced with undiluted chlorhexidine rinse, concentrated coffee and tea for a period of 23 days. After staining, the teeth were examined by ESEM, then brushed using a toothbrushing machine. Imaging was repeated after 5, 10, 15 and 30 seconds of brushing. As seen with ESEM, the Arm & Hammer product had different effects than those from the distilled water control, suggesting something other than that expected from abrasive and mechanical forces alone. There were also differences from the Crest dentifrice removal on this single sample, suggesting a possible difference between the two products. Further studies are needed to confirm and explain these effects.

Chlorhexidine↗
PubMed2004

Endotoxin measurement in house dust using the end-point Limulus amoebocyte lysate method.

Endotoxin is a lipopolysaccharide, a part of gram-negative bacteria cell membrane commonly present in general and many occupational environments. This paper describes sample preparation and endotoxin measurement in 16 samples of house dust from urban homes (Zagreb, Croatia) using end-point chromogenic Limulus amoebocyte lysate (LAL) bioassay. House dust was collected on cellulose filters by vacuuming bedroom and living room floors, and was kept frozen until assayed. Samples were extracted from filters with a 0.05% solution of Tween-20 in endotoxin-free water. Serial dilutions of samples were measured in duplicates. The linearity of the standard curve was satisfying (r=0.983), as well as the recovery (92 and 110%) and repeatability (coefficient of variation from 0 to 8.5%). The endotoxin levels found in the house dust samples ranged from 4.8 to 200 EU/mg, with the arithmetic mean of 49.5 EU/mg (standard error of the mean of 12.1 EU/mg), and were in the range of house dust endotoxin values obtained by other authors.

Air Pollution, Indoor↗
PubMed1986

A review of computer ECG analysis: time to evaluate and standardize.

The use of computers for the interpretation of the resting electrocardiogram (ECG) is critically reviewed. After a short description of the motives for the application of computers in this field, the processes that are sequentially executed in an ECG data processing system are critically analyzed, i.e., the acquisition and transmission of signals; the wave recognition and measurement phase; the parameter extraction, rhythm analysis, and classification phases; and, finally, the reporting of results as well as serial comparison. Emphasis is given on the evaluation and standardization of measurement and interpretation results of various ECG computer programs, based on the experience of the author as project leader of the International Cooperative Project "Common Standards for Quantitative Electrocardiography" (CSE). An extensive list of references is provided.

Adult↗
PubMed1994

RME-induced root resorption and repair: a computerised 3-D reconstruction.

This study was designed to evaluate the 3-D relation of epithelial rests of Malassez (ERM) to orthodontically induced resorption lacunae in human tooth roots. First premolars were extracted following rapid maxillary expansion, then they were prepared for transmission electron microscopy and sectioned. Serial 1 micron sections were used for the 3-D reconstructions and ultra thin sections were utilized to verify the identification of epithelial cells and blood vessel types. Each serial section was photographed and profiles of the resorption bays, epithelial cells and blood vessels were digitized and computer reconstructions performed with an IBM-based software programme. The reconstructions supported the view that epithelial cell and blood vessel relation to the resorption lacunae appeared to be related to the level of activity of the bay. Possible functional roles for epithelial cells in the resorptive process are discussed.

Adolescent↗
PubMed2006

Serial gene expression profiling in the intact human heart.

BACKGROUND: In chronic heart failure due to a dilated cardiomyopathy phenotype, the molecular bases for contractile dysfunction and chamber remodeling remain largely unidentified. METHODS: To investigate the feasibility of measuring global gene expression serially in the intact failing human heart, we performed repeated messenger RNA (mRNA) expression profiling using RNA extracted from endomyocardial biopsy specimens and gene chip methodology in 8 subjects with idiopathic dilated cardiomyopathy. In patients treated with beta-blocking agents or placebo, myocardial gene expression was measured in endomyocardial biopsy material and radionuclide ejection fraction was measured at baseline and after 4 to 12 months of treatment. Gene expression was measured for 12,625 gene sequences by using Affymetrix U95 gene chips and commercially available software. For 6 mRNAs, gene chip results were compared with measurements made by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In an unfiltered composite analysis of changes in expression detected in the patients with high-signal intensity chips, 241 genes showed an increase and 331 genes a decrease in mRNA abundance. There was good agreement between changes measured by quantitative RT-PCR and those determined by gene chips. There was less variance between differences in phenotype in patients sampled serially as compared between subjects with similar phenotypes sampled at baseline. CONCLUSIONS: Serial gene expression profiling with association to phenotypic change is feasible in the intact human heart and may offer advantages to cross-sectional expression profiling. This study suggests that the intact failing remodeled human heart is in an activated state of gene expression, with a large net reduction in gene expression occurring as phenotypic improvement occurs.

Adolescent↗
PubMedDate not supplied

Robotic solid phase extraction and high performance liquid chromatographic analysis of ranitidine in serum or plasma.

A fully automated assay for the analysis of ranitidine in serum and plasma, with and without an internal standard, was validated. It utilizes robotic solid phase extraction with on-line high performance liquid chromatographic (HPLC) analysis. The ruggedness of the assay was demonstrated over a three-year period. A Zymark Py Technology II robotic system was used for serial processing from initial aspiration of samples from original collection containers, to final direct injection onto the on-line HPLC system. Automated serial processing with on-line analysis provided uniform sample history and increased productivity by freeing the chemist to analyse data and perform other tasks. The solid phase extraction efficiency was 94% throughout the assay range of 10-250 ng/mL. The coefficients of variation for within- and between-day quality control samples ranged from 1 to 6% and 1 to 5%, respectively. Mean accuracy for between-day standards and quality control results ranged from 97 to 102% of the respective theoretical concentrations.

Biological Availability↗
PubMed2000

[Quick RNA extraction from specified morphological region in frozen sections and its RT-PCR application].

OBJECTIVE: To establish a method for quick RNA extraction from specified histological region in frozen sections so as to avoid cross RNA contamination among different types of cells/tissues and to achieve specific RT-PCR amplification. METHODS: Prepare serial frozen sections 5microm in thickness for histological and immunocytochemical examination. Based on the results, make one or two pieces of 20 microm section and select a specified region for quick RNA extraction and RT-PCR analysis. RESULTS: RNA extraction can be finished within an hour and the data of RT-PCR obtained within 6 hours. Besides the high performance, this method can efficiently exclude pseudo positive or pseudo negative results caused by inter-cellular RNA contamination. CONCLUSION: This method is an important complement to the current techniques for RNA extraction and RT-PCR.

Frozen Sections↗
PubMed1998

Double-blind, placebo-controlled evaluation of sublingual immunotherapy with standardized olive pollen extract in pediatric patients with allergic rhinoconjunctivitis and mild asthma due to olive pollen sensitization.

For evaluation of the efficacy and the safety of specific sublingual immunotherapy with high allergen dose, 66 children with seasonal asthma, rhinitis, and conjunctivitis due to sensitization to olive pollen were enrolled in a double-blind, randomized, placebo-controlled study between October 1994 and October 1996 in Greece. Thirty-four patients were randomly allocated to the active group, and 32 received placebo. Immunotherapy consisted of olive-allergen extracts (Stallergènes SA) administered sublingually pre- and coseasonally from January to July for 2 consecutive years. Serial concentrations from 1 to 300 IR. were used up to the maintenance dose of 20 drops of 300 IR daily. The cumulative dose for each patient was 300 times higher than in parenteral immunotherapy, and the cumulative dose of the major allergen Ole e 1 was 8.1 mg/2 years. The patients were assessed by clinical parameters (symptom and medication scores from patients' daily diaries) and immunologic measurements (specific IgE, IgG4, eosinophil cationic protein [ECP]) were performed. The actively treated patients had a significantly lower score for dyspnea (P<0.04 during the first season; P<0.03 during the second season). At the pollinic peak during the second year, a lower score of conjunctivitis was recorded (P<0.05) in the actively treated patients. The analysis of intragroup evolution showed that the total score of rhinitis increased significantly during the pollinic peak in the group under placebo, whereas there was no symptomatic peak for the same period in the group under active treatment. However, the difference between the groups was not significant. The medication score did not differ significantly between the groups. Oral steroids were the only variables with a P value near the significance level (P=0.06) in favor of the actively treated group. A significant decrease in skin reactivity was recorded in the active group after 2 years of treatment. No significant variation in specific IgE and IgG4 was detected. A significantly lower level of serum ECP was observed at the pollinic peak in the actively treated patients during the first pollen season (P=0.01), but this was not confirmed the second year when the ECP levels doubled in both groups without correlation to the clinical findings. Tolerance was excellent with only a few minor side-effects reported. In conclusion, high-dose specific sublingual immunotherapy appears to be safe and effective in improving mild seasonal asthma and conjunctivitis linked to olive-pollen sensitization.

Administration, Sublingual↗
PubMed2006

Detection and differentiation of Bordetella spp. by real-time PCR.

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.

Bordetella↗