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PubMed1996

[New sources of lectins to differentiate between Trypanosoma cruzi y T. rangeli].

Extracts of 176 species of Colombian plant seeds, corresponding to 49 families and 147 genera, were tested for detecting agglutinins against human red blood cells from A+, B+ and O+ groups, dog, horse and rabbit. Extracts with haemagglutination activity were used for agglutination of Trypanosoma cruzi and T. rangeli. In addition the hemolymph of 16 native species of invertebrates were tested in the same conditions. Serial dilution of extracts were used for agglutination reactions. Both T. cruzi and T. rangeli epimastigotes showed agglutination with the extract of seven different species of plant seeds and with two types of haemolymph of invertebrates. The seeds of five plants exclusively agglutinated the epimastigotes of T. cruzi and thus can be used for the differentiation between culture forms of the trypanosomes. The secretion of the lung of a snail (Bulimus sp.) lysed entirely the epimastigotes of T. cruzi but did not affect T. rangeli forms. No extracts were found which agglutinated or lysed exclusively the epimastigotes of T. rangeli.

Animals↗
PubMed1993

Antigestagenic activity of Ixora finlaysoniana in rat.

Oral administration of crude ethanolic extract of the serial parts of Ixora finlaysoniana Wall. ex G. Don to adult female rats at 250 mg/kg dose on days 1-5 or 1-7 post-coitum prevented pregnancy in 100% rats. The extract was also effective when administered on days 1 or 1-3 post-coitum, but the minimum effective dose increased with decreased duration of administration and was 1000 mg/kg and 500 mg/kg, respectively, in the two schedules. At lower doses, a significant reduction in implantation number and increased post-implantation resorption rate were observed in all the schedules. Almost complete resorption of all implantations was observed after administration of 1000 mg/kg dose of the extract during the peri-implantation period. A slight acceleration in tubal transport rate of embryos and delay in blastocyst formation were observed in rats treated postcoitally with the single anti-implantation dose of the extract. Significantly fewer embryos were recovered after their entry into the uterus. Except in one rat receiving 250 mg/kg dose of the extract on days 1-5, in which one apparently normal zona-free blastocyst was recovered from the uterus, uterine flushings of none of the nonpregnant animals contained any unimplanted embryos by day 10 post-coitum. In immature rat bioassay, the extract was found to possess estrogenic activity as evidenced by dose-dependent increase in uterine weight and cornification of the vaginal epithelium at doses ranging from 50-1000 mg/kg. At the 500 and 1000 mg/kg doses, it also induced premature opening of the vagina. Taking 100% increase in uterine weight as the parameter, the extract was found to be about 1.6X10(5) times less estrogenic than ethinylestradiol. The extent and duration of estrogenic responses exerted by single contraceptive dose of the extract were also markedly lower than that induced by ethinylestradiol. The extract was devoid of any estrogen antagonistic or synergistic activity and did not affect ovarian prenidatory estrogen or progesterone synthesis. The findings indicate that the extract at its contraceptive dose a) exerts a differential estrogenic response at the fallopian tube and the uterine levels, b) does not appear embryocidal, but causes slight asynchrony in development and tubal transport rate of pre-implantation embryos, which together with their loss through vagina after entry into the uterus, due to estrogenic action of the extract, might contribute to its anti-implantation action, and c) its anti-implantation and post-implantation resorptive actions are not mediated via altered ovarian function.

Animals↗
PubMed1977

Effect of soluble extracts from periodontal dressings on human granulocytic leukocytes in vitro.

Purified populations of human polymorphonuclear granulocytic leukocytes were exposed in vitro to solubilized material(s) extracted from two commerical periodontal dressings. The composiitonof one dressing contained eugenol while that of the other lacked it. Extracts were prepared by placing each mixed dressing in isotonic saline for 18 to 24 hours. Granulocytic leukocytes received serial dilutions of each extract and were then evaluated at 15 minutes and at 3 hours of culture for changes in viability and elaboration of cyto-plasmic and lysosomal enzymes. High concentrations of extract from each dressing were toxic to the cultured granulocytes. Measurements for cell viability and release of cytoplasmic enzyme revealed that the toxic potential in the eugenol-free dressing was greater. With cell damage, extracellular release of the lysosomal enzymes was found to occur. Serially increased dilutions of either extract to nontoxic concentrations failed to selectively release lysosomal enzymes.

Acid Phosphatase↗
PubMed2006

Estimating amplification efficiency improves multiplex real-time PCR quantification of Bacillus licheniformis and Bacillus subtilis spores in animal feed.

A multiplex real-time PCR assay was developed for absolute quantification in animal feed of Bacillus subtilis CH201 and Bacillus licheniformis CH200 spores, which constitute the viable component of the microbial growth promoter, BioPlus 2B. Spores were lysed using a bead-beating protocol. DNA was extracted and purified from the lysates with the Qiagen DNeasy Plant Kit. Two standard curves for absolute quantification were made and tested. Standard curve-1 was made from feed samples spiked with BioPlus 2B, while standard curve-2 was made from serially diluted DNA extracted from BioPlus 2B powder. Feed samples supplemented with BioPlus 2B were quantified using both standard curves. The detection limit of the assay was 10(4) CFU g(-1) of feed. The amplification efficiency (Eff) of each PCR was determined using the LinRegPCR software and Eff differences between individual samples and standards were corrected for. When compared to plate counts, standard curve-1 slightly under-estimated the number of spores (mean=-2.47% of plate counts). A spore density-dependent Eff was found, and Eff for standard curve-1 could not be determined. Standard curve-2 over-estimated spore numbers when not corrected for individual Eff (mean=+5.46% of plate counts). Standard curve-2 Eff was independent (Eff(mean)=1.96) of spore density. The assay quantified the numbers of spores in feed samples very similar to plate counts (mean=+0.47% of plate counts), when standard curve-2 was used and individual Eff was accounted for.

Animal Feed↗
PubMed1980

Ectopic production of methionine enkephalin and beta-endorphin.

Immunoreactive methionine enkephalin and beta-endorphin were sought by serial dilution of tissue extracts and assay of chromatographic fractions in non-endocrine tumour tissue from three patients with the ectopic adrenocorticotrophin syndrome associated with carcinoid tumours and in normal lung tissue and thymic tissue from a patient with myasthenia gravis. In all cases serial dilution of extracts showed parellelism to standard radioimmunoassay curves. The two peptides were found in high concentration in the three tumours but were undetectable in the control tissues. In a single case tested the methionine enkephalin concentration in a vein draining the tumour was twice that in a peripheral vein. In view of their profound effect on behaviour in animals and potent analgesic activity in animals and man the ectopic secretion of methionine enkephalin and beta-endorphin may modify the clinical features of a wide variety of tumours and produce some of the diverse clinical syndromes associated with malignancy.

ACTH Syndrome, Ectopic↗
PubMed2005

Is the use o f Gunnera perpensa extracts in endometritis related to antibacterial activity?

Rhizome extracts of Gunnera perpensa are used in traditional remedies in South Africa to treat endometritis both in humans and animals. An investigation was undertaken to determine whether this plant possesses antibacterial activity, which may explain its efficacy. Gunnera perpensa rhizome extracts were prepared serially with solvents of increasing polarity and tested for antibacterial activity. Test bacteria included the Gram-positive Enterococcus faecalis and Staphylococcus aureus and the Gram-negative Escherichia coli and Pseudomonas aeruginosa. A moderate to weak level of antibacterial activity in most of the extracts resulted, with the best minimal inhibitory concentration (MIC) value of 2.61 mg ml(-1) shown by the acetone extract against S. aureus. The extracts were also submitted to the brine shrimp assay to detect possible toxic or pharmacological effects. All the extracts were lethal to the brine shrimp larvae at a concentration of 5 mg ml(-1). The acetone extract was extremely toxic at 1 mg ml(-1), with some toxicity evident at 0.1 mg ml(-1). The remainder of the extracts generally displayed little activity at concentrations lower than 5 mg ml(-1). In summary, the results indicate that although the extracts demonstrated a level of pharmacological activity, the relatively weak antibacterial activity is unlikely to justify the use of G. perpensa rhizomes in the traditional treatment of endometritis. Rather, the slightly antibacterial nature of the rhizomes may contribute to an additive effect, along with their known uterotonic activity, to the overall efficacy of the preparation.

Animals↗
PubMed1980

A new extraction of arginine vasopressin from blood: the use of octadecasilyl-silica.

Small columns packed with octadecasilyl-silica were used to extract arginine-vasopressin in femtomole amounts from biological fluids for radioimmunoassay. This method is effective in isolating the peptide from substances in plasma and serum which interfere with its quantitation. Consistent and reproducible results and recoveries > 80% were obtained with this procedure. High pressure liquid chromatography and serial dilution of extracted samples confirm the identity of the extracted product.

Animals↗
PubMed1982

Extraction procedures for oilseeds and related high fat-low moisture products.

A combined sample preparation/extraction procedure is presented for pesticide residue analysis of oilseeds and related high fat-low moisture products. The procedure utilizes high-speed milling to prepare the sample and high-speed homogenization in the extraction step to achieve what is apparently quantitative isolation of both incurred residues and natural oils. A separate, simple, oil determination step allows findings to be reported on either the fat or whole product basis. Petroleum ether, ethyl ether-petroleum ether (1 + 1), and ethanol are used serially as the extractants. Usual fatty food cleanup procedures and multiresidue gas chromatographic detection techniques are utilized. The procedure presented in this paper is a refinement of earlier work which used a homogenizer both to grind and to extract samples of unground seeds and which demonstrated essentially complete extraction of endrin residues in soybeans and DDT residues in mustard seed. Identical samples analyzed by the currently recommended shakeout procedure, 29.012, gave recoveries of approximately 50% of the total residues. The procedure presented in this paper was satisfactorily tested on 13 different oilseed types and one sample of soda crackers. Oil content for these samples ranged from 5 to 69%.

Arachis↗
PubMed1984

Varying bioactive to immunoactive ratios of the human chorionic gonadotropin-like substance present in normal human tissues.

To further characterize the hCG-like substance found in normal nonpregnant human tissues, we compared the biological (B) and immunological (I) activities of the substance in extracts of 40 nontrophoblastic tissues and 7 placentas. B was measured in an in vitro mouse testicular interstitial cell bioassay, and I was measured in the beta hCG RIA. Highly purified hCG (CR 119) was used as a standard. B was detected in 92% of the nontrophoblastic nonpituitary tissues. Parallelism between serial dilutions of extracts and the hCG standard was found for 75% of the pituitary extracts, 86% of the placental extracts, and 53% of the other tissues. The correlation between B and I activities in the nontrophoblastic nonpituitary tissues was good (r = 0.8; P less than 0.001). Preincubation of the tissue extracts with anti-hCG serum before bioassay significantly reduced the B activity. There was wide variation in the B to I ratio between tissues and within the same tissues from different individuals. A B to I ratio less than 1 was found in 50% of the tissues, excluding placenta and pituitary, and may have been due to the presence of incomplete or altered hCG molecules, which are immunologically active but have reduced B activity.

Animals↗
PubMed1990

Spinal cord thromboplastin-induced coagulopathy in a rabbit model.

Coagulopathy results from many diverse events, including several neurogenic causes. Using a rabbit model, we produced coagulopathy by injecting autologous spinal cord and extracted thromboplastin intravenously. Serial coagulation panels were performed to evaluate the activation of the thrombotic and fibrinolytic pathways. Group 1 animals (n = 4) received intravenous injections of homogenized spinal cord tissue. Coagulopathy was not produced with 36 mg of homogenized spinal cord tissue, but 50 mg or more resulted in death. Group 2 animals (n = 12) received intravenous injections of extracted rabbit cord thromboplastin, which contained approximately 60% activity of a commercially purified rabbit brain thromboplastin. Five animals receiving 2.5 to 5.5 mg of thromboplastin per kilogram of body weight survived with evidence of coagulopathy. Seven animals receiving 2.5 to 100 mg of thromboplastin per kilogram of body weight died. Group 3 (4 control animals) received normal saline injections without changes in clinical or laboratory status. The thrombotic pathway was activated in all animals as evidenced by decreased platelet counts and fibrinogen levels. Activation of the fibrinolytic system was demonstrated by increased concentrations of protamine sulfate and abnormal euglobulin clot lysis times. The most sensitive parameters were the platelet count, protamine sulfate concentration, and white cell count (margination), which became abnormal within 15 minutes after the injections and returned to normal within 1 hour.

Animals↗
PubMed1997

Species-specific polymerase chain reaction for the differentiation of larvae from Dictyocaulus viviparus and Dictyocaulus eckerti.

Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10,000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.

Animals↗
PubMed1979

Thyrotropin-releasing hormone activity in the human placenta.

TRH immunological and biological activities were found in extracts of human placentas. Eight term placentas delivered by cesarean section were extracted with 2 N acetic acid, followed by glacial acetic acid. Lyophilized samples were resuspended in phosphate-buffered saline. TRH activity, identified in each placental extract by specific RIA, averaged 19.8 +/- 3.3 pg/mg protein. TRH in the placental extracts was similar to synthetic TRH by four criteria. 1) Serial dilutions of placental extracts defined an immunoassay inhibition curve with a slope (-1.93) identical to synthetic TRH (-1.88). 2) When placental extract or synthetic TRH was chromatographed on Sephadex G-10, TRH immunoreactivity was found in similar fractions of the eluate. 3) Both placental extract and synthetic TRH stimulated TSH release from rat pituitaries in vitro. 4) Human serum degraded placental extract and synthetic TRH in a qualitatively similar manner. Placental minces did not accumulate exogenous [3H]TRH, suggesting that the placental TRH may originate endogenously.

Animals↗
PubMed2004

Dose dependence and time course of the immunologic response to administration of standardized cat allergen extract.

BACKGROUND: The immunologic response to allergen immunotherapy with 3 serial 5-fold doses of cat extract has been studied after approximately 5 weeks of immunotherapy. The highest dose containing 15 mug of Fel d 1 produced the most consistent and favorable response. It is unknown whether the comparative response on reaching a maintenance dose is maintained with long-term maintenance therapy. OBJECTIVE: The purpose of this investigation was to evaluate the immunologic responses with these 3 serial doses of cat hair and dander extract at baseline, after reaching the maintenance dose (approximately 5 weeks), and after 1 year of maintenance immunotherapy. METHODS: Twenty-eight patients with cat allergy randomized in a double-blind study were assigned to one of 4 treatment groups: placebo or cat hair and dander extract containing 0.6 mug of Fel d 1, 3 mug of Fel d 1, and 15 mug of Fel d 1 at maintenance. Studies included skin prick tests and late cutaneous reactions with cat hair and dander extract, titrated nasal challenges with the extract, serum cat allergen-specific IgG4 and IgE measurement, and flow cytometric and ELISA analysis of whole blood and intranasal cytokines (TGF-beta, IL-10, IFN-gamma, IL-4, and IL-5). RESULTS: Twenty-six subjects completed the study. After both 5 weeks and 1 year, significant and dose-dependent differences were seen with total symptom scores on nasal challenge ( P < .0001), with titrated skin prick testing with cat dander extract at 5 weeks ( P = .014) and 1 year ( P < .0001), and with cat-specific IgG4 measurement at 5 weeks ( P = .004) and 1 year ( P = .003). At 1 year, neither flow cytometry of whole blood nor ELISA evaluation of nasal cytokines demonstrated any significant differences among the treatment groups. CONCLUSION: The response to titrated nasal allergen challenge, titrated skin prick testing, and allergen-specific IgG4 measurement to cat immunotherapy at 5 weeks is predictive of the response at 1 year.

Allergens↗
PubMed2003

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects.

BACKGROUND: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. METHODS: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. RESULTS: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were < or =12% and < or =10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 +/- 0.26 fmol/ml (mean +/- SD). CONCLUSIONS: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1.

Animals↗
PubMed1978

Radioimmunoassays for prostaglandins. I. Technical validation of prostaglandin F2alpha measurements in human plasma using sephadex G-25 gelfiltration.

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F2alpha (PGF2alpha). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid or Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/BO 0.9-0.2). Upon comparison of separation by Polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF2alpha is 97.6%.

Chromatography, Gel↗
PubMed1993

Prolonged increases in neurotrophic activity associated with kainate-induced hippocampal synaptic reorganization.

Synaptic reorganization occurs in the hippocampus following various forms of seizure activity and injury, and may contribute to epileptogenesis. To address the hypothesis that neurotrophic factors play an inductive role in synaptic reorganization following seizures, we directly measured neurotrophic activity in rat hippocampal extracts after kainate injection or prolonged stimulation of the perforant path. Serial dilutions of hippocampal extracts were added to cultures of chick dorsal root ganglia, which are known to require trophic support from nerve growth factor and other neurotrophins, or ciliary ganglia neurons, which require trophic support from ciliary neurotrophic factor. Neurotrophic activity was significantly increased in hippocampal extracts harvested from 12 h to 2 months after kainate treatment, with the peak effect seen at seven days. This neurotrophic activity was substantially blocked by an anti-nerve growth factor antibody. Extracts at seven days also showed a significant increase in ciliary neurotrophic factor-like activity. Sulfide/silver histochemistry, which stains dentate granule cell axon terminals, revealed that mossy fiber sprouting was evident two weeks following kainate treatment and increased progressively over the next two to six weeks. Perforant path stimulation that produced hyperexcitability in the dentate gyrus, but no sprouting, failed to induce changes in neurotrophic activity. These results suggest there are significant increases in neurotrophic factors following kainate-induced seizures, and the increases may be related to kainate-induced hippocampal injury rather than seizures per se. Furthermore, the timecourse of increased neurotrophic activity parallels that of mossy fiber reorganization, and is consistent with the hypothesis that neurotrophic factors play a role in the injury-induced synaptic reorganization seen in epilepsy.

Animals↗
PubMed1979

Letter detection with rapid serial visual presentation: evidence against word superiority at feature extraction.

Letter detection typically is faster and more accurate in words than nonwords. Experiments 1, 2, and 3 tested the robustness of the word superiority effect using rapid serial visual presentation of words or nonwords. Letter detection was better in words even when the six-letter items were presented one after the other at rapid rates, up to about 10 items per second. At yet faster rates, however, the word advantage vanished. Experiments 4 and 5 tested whether word context aids feature extraction or the subsequent interpretation stage. In Experiment 4, subjects had to discriminate whether a mutilated A or mutilated E was present; in Experiment 5, subjects had merely to decide whether a mutilated A was present. Mutilation discrimination in Experiment 4 was better on words than nonwords; once a mutilation was detected, the word context revealed whether it was an A or an E. Mutilation detection in Experiment 5 did not differ between words and nonwords, though on words there was a response bias toward not reporting a mutilation as present. The results indicate that familiarity aids the interpretation process alone: Letters are not seen any more clearly or rapidly in words, but are simply filled in or inferred more accurately from the familiar context.

Discrimination Learning↗
PubMed1984

Direct visualization of particle velocity distribution by pseudostereoscopic viewing of time-lapsed sequential images: application to fast axonal transport.

We describe a simple method for direct visualization of the velocity distribution of particles moving against an immobile background. The technique involves pseudostereoscopic viewing of image pairs separated by an appropriate time interval in a sequential recording of the subject. Under these conditions, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement, and with the degree of perceived elevation being proportional to its speed. In effect, the binocular optic axis becomes a velocity (speed) axis under these conditions. The technique is illustrated with examples of image pair sequences showing fast axonal transport in lobster and squid axons using video-enhanced differential interference contrast microscopy. However, the pseudostereoscopic method is quite generally applicable to both microscopic and macroscopic time-dependent phenomena. Particle speeds can be quantitated using standard procedures for measuring frame-to-frame particle displacements, or alternatively, by determination of parallax using stereogrammatic methods. It should be also readily adaptable for on-line monitoring of particle velocity distribution, particularly in video systems where frame buffers can be utilized to extract and present serial image pairs having any desired time separation from video-taped sequences.

Animals↗