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PubMed1984

Oxytocin content of human fetal pituitary glands.

Seventeen human fetal and neonatal pituitary glands were removed at necropsy, and analyzed for oxytocin content by a specific and sensitive radioimmunoassay, after homogenization in 0.4M acetic acid. Serial dilution of the pituitary extract showed parallelism with the oxytocin standard curve on the radioimmunoassay. Column chromatography of the pituitary gland extract gave a single peak of immunoreactive oxytocin as determined by the radioimmunoassay. In eight fetuses, 14 to 17 weeks' gestation, pituitary gland oxytocin was 10.2 +/- 5.9 ng/gland (mean +/- SE), whereas an 18-week fetus had 5.8 ng of oxytocin per gland. Pituitary gland oxytocin content increased to 38.4 ng/gland in a 20-week fetus removed at hysterectomy, and 31.6 ng/gland in a 26-week fetus. Fetal pituitary gland oxytocin values were 22.1 ng/gland and 57.0 ng/gland at 32 weeks and increased significantly to 544.3 +/- 33.8 ng/gland in 1- to 5-day-old term newborn infants (n = 3). However, a 13-day-old term newborn infant had 3.7 ng of oxytocin per pituitary gland. The increased pituitary gland oxytocin content with advancing gestation was due to a significant increase in oxytocin concentration rather than to an increase in the weight of the pituitary gland. The findings indicate that oxytocin is present in fetal pituitary glands as early as 14 to 17 weeks' gestation and increases at term to 50 and 13 to 14 times more than in early midtrimester and early third-trimester pregnancy, respectively.

Chromatography, Gel↗
PubMed1984

De novo synthesis and axoplasmic transport of [35S]methionine-substance P in explants of nodose ganglion/vagus nerve.

The synthesis and transport of substance P, the widely distributed undecapeptide, was studied in the vagus nerve of the guinea pig. In preliminary in vivo studies, the cervical vagus nerve was ligated 2 cm distal to the nodose ganglion. Twenty-four hours later, the content of immunoreactive substance P (IR-SP) in the 3-mm nerve segment proximal to ligature was 2147 +/- 207 pg (mean +/- S.E.M.) vs 133 +/- 31 pg in an equal segment of unligated nerve or 243 +/- 55 pg in the nodose ganglion. When the vagus nerve was crushed above the ganglion and simultaneously ligated 2 cm distally, the IR-SP content proximal to the ligature was reduced 50% to 1131 +/- 99 pg (P less than 0.01), while nodose ganglion content increased to 420 +/- 140 pg (n.s.). To confirm that residual transport following supranodose crush was derived from nodose ganglion-synthesized SP, SP synthesis and transport were studied in explants of nodose ganglion and attached distal vagus nerve removed from perfused animals and maintained in vitro for up to 24 h. At the time of resection, nerves were ligated 1.5 cm distal to the ganglion. Twenty-four hours following explantation, IR-SP content in proximal segments was 1022 +/- 142 pg vs 155 +/- 22 pg in unligated segments and 560 +/- 72 pg in the nodose ganglion. Accumulation in the proximal segment was time dependent. In separate experiments, [35S]methionine was added to explant medium and the explants maintained for varying time intervals. Nerve tissue was extracted and subjected to either serial reverse phase high performance liquid chromatography (HPLC), or immunoprecipitation with SP antiserum followed by a single HPLC separation. By 4 h, radiolabeled SP was present in nodose ganglia and lesser amounts in the proximal segments. By 12 h, [35S]SP was present equally in ganglia and proximal segments whereas by 18 h, two-thirds or more of the newly synthesized peptide was present in proximal segments. At 18 h, the quantity of radiolabeled SP covaried with IR-SP content in the individual nerve segments. The addition of cycloheximide to explant medium reduced [35S]SP synthesis by 90%. These studies demonstrate that: (1) approximately 50% of immunoreactive SP transported efferently within the vagus nerve of the guinea pig is derived from the nodose ganglion, (2) de novo SP synthesis within and export from the nodose ganglion occurs within 4 h, (3) the changes in IR-SP content demonstrated in in vivo and in vitro ligation studies accurately reflect ongoing SP synthesis within the nodose ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

The use of mosquitoes (Aedes aegypti) to detect ciguatoxin in surgeon fishes (Ctenochaetus striatus).

A new animal assay to detect ciguatoxin in fishes was developed. Mosquitoes were subjected to intrathoracic injection of serial dilutions of ciguatoxin crude extracts. Toxicity of the fishes was expressed as the mosquito LD50 (g of flesh per mosquito). A significant correlation between the mosquito bioassay and the mouse bioassay performed on the same extracts was shown (r = 0.72, n = 80). We were able to detect 96% of the toxic fishes, while 91% of the fishes non-toxic in mice were also non-toxic in mosquitoes.

Aedes↗
PubMed1989

Circadian variation of skin reactivity and allergy skin tests.

Previous investigations of the circadian variation in skin reactivity suggested that results of skin tests obtained in the afternoon could vary from the results obtained in the early morning and therefore could result in a differing assessment of patient sensitivity. To determine whether this was a practical concern in the normal clinical setting, we studied 20 adults and 20 children who had skin prick tests positive (3+ or more) to short ragweed. These patients were skin tested in duplicate at 8 AM and at 4 PM with fivefold serial dilutions of short ragweed extracts (1:20 to 1:12,500, wt/vol) and of histamine hydrochloride (10 to 0.016 mg/ml). Areas of wheal and flare were recorded and measured by computed planimetry. In addition, results were also read according to a conventional scoring system. Mean wheal and erythema areas with ragweed and histamine at each dilution were compared between morning and their corresponding evening values. Although there was a trend for the morning means to be larger than evening means, no significant differences between the two sessions were observed at any dilution. Mean morning skin index scores, as calculated from the combined mean wheal and erythema areas, were larger than mean evening scores for ragweed and histamine, but the differences were not of a degree to be clinically important. This observation was also true for conventional scores. Comparing the results from the two groups of children who had their first set of skin tests performed either in the morning or afternoon session indicated that there was no evidence of a refractory state of the skin during the second test sessions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMedDate not supplied

Localization and characterization of the N-terminal fragment of atrial natriuretic factor (ANF) precursor in the frog heart.

The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.

Animals↗
PubMedDate not supplied

Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system.

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.

Animals↗
PubMed1993

Immunoreactive atrial natriuretic peptides in the oocyte.

1. The presence and partial characterization of immunoreactive ANP (irANP) in eggs were investigated using high performance liquid chromatography (HPLC), immunohistochemistry and Northern-blot hybridization in mammalian and nonmammalian vertebrates. 2. Serial dilution curves of egg extracts from frogs and freshwater teleostean fishes (silver crusian carp and loach) were parallel to the standard curve of atriopeptin III. Rat oocytes also contained irANP (25-30 pg/egg). 3. The HPLC profile of irANP showed two main peaks corresponding to low and high molecular weight of irANP. 4. ANP mRNA was detected in the fish eggs. With immunohistochemical analysis of the rat ovarian follicles, this ANP-like material was localized mainly at the oocytes. 5. We, therefore, suggest that the vertebrate oocytes synthesize ANP and that the presence of ANP in the oocytes may be linked with the cell differentiation.

Animals↗
PubMed1988

Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation and gonadectomy.

A heterologous inhibin radioimmunoassay (RIA) method has been developed which is highly specific and of sufficient sensitivity to detect inhibin in female and male rat serum. Purified bovine 31 kDa inhibin was used in the generation of the antiserum and following iodination as tracer in the assay. Parallel logit-log dose-response lines were observed between a rat ovarian extract used as standard and serial dilutions of female and male serum and testicular interstitial fluid. The within-assay variation based on index of precision was 0.049 (n = 5) and the between-assay variation (n = 4) was 9.8%. The specificity of the assay was assessed from: (a) the failure of a number of structurally related proteins (activin-A, transforming growth factor-beta (TGF-beta), Müllerian inhibitory substance) as well as inhibin subunits to crossreact (less than 0.5%) in the assay relative to bovine 31 kDa inhibin; (b) nondetectable levels of immunoactivity in the serum of gonadectomised rats; and (c) a close correlation (r = 0.96) between serum levels of in vitro biological and immunological activities from rats following pregnant mare serum gonadotropin (PMSG) stimulation. Similar initial t 1/2 values (14-15 min) of serum inhibin following gonadectomy were obtained in both sexes. This RIA method will be useful in the study of the physiology of inhibin in the female and male rat.

Animals↗
PubMed1989

Development of an avian calcitonin radioimmunoassay using synthetic chicken calcitonin as immunogen.

1. A sequential double antibody radioimmunoassay (RIA) has been developed using synthetic chicken calcitonin (CT) as antigen, tracer and standard. 2. The immunoassay has a minimum detection limit of 0.5 ng and effective dose (ED50) of 7 ng. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of CT standard. Extracts of chicken and turkey ultimobranchial glands caused parallel displacement of tracer similar to synthetic CT. 3. Primary antisera (anti-chicken CT) was raised in guinea pigs immunized with RIBI: animals treated with Freund's complete adjuvant failed to respond. 4. Chicken CT was determined to have a half-life of 60 sec in the turkey hen. Development of a homologous RIA for avian CT will allow studies to elucidate the role of this hormone in birds.

Animals↗
PubMed1994

Pharmacokinetics of the three radioiodinated dopamine D2 receptor ligands [123I]IBF, [123I]epidepride and [123I]2'-ISP in nonhuman primates.

The pharmacokinetics of three radioiodinated high affinity dopamine D2 receptor binding ligands-two benzamide neuroleptics (IBF and epidepride) and the butyrophenone neuroleptic analog 2'-iodospiperone (2'-ISP)-were measured in nonhuman primates. [123I]IBF and [123I]epidepride were prepared by iododestannylation of the corresponding tributylstannyl derivative, whereas [123I]2'-ISP was labeled by (NH4)2SO4 mediated iodide for bromide exchange starting from 2'-bromospiperone. Labeled products were purified by HPLC and were obtained in > 93% radiochemical purity and > 7000 Ci/mmol sp. act. After i.v. injection in baboons, serial arterial plasma samples were extracted with ethyl acetate (IBF and epidepride) or denatured with methanol (2'-ISP) and analyzed by HPLC. For IBF, plasma levels of parent compound dropped to 50% of the plasma activity within 20 min post injection and the major radiometabolite was lipophilic. For epidepride, it took 30-40 min for parent content to reach 50% and the major radiometabolite was polar. For 2'-ISP, parent composition dropped to 60% after about 15 min. Arterial input curves for IBF and epidepride fit three-exponential models with terminal half-life of 54-76 and 50-59 min, respectively. Whole body images were acquired using the conjugate counting method. The distribution of all three agents was qualitatively similar, with major excretion through the hepatobiliary route. Peak whole brain uptake, observed within 20 min for all three tracers, was estimated as 7% injected dose for [123I]IBF, 8% for [123I]epidepride and 5% for [123I]2'-ISP.

Animals↗
PubMed2005

In vitro and in vivo effects of isolated fractions of Brazilian propolis on caries development.

Recently, two chemically different types of Brazilian propolis (type-3 and -12) were shown to have cariostatic properties. This study aimed to evaluate the influence of their isolated fractions on mutans streptococci viability, glucosyltransferases (GTFs) activity and caries development in rats. The ethanolic extracts of propolis (EEPs) were serially fractionated into hexane (H-fr), chloroform, ethyl acetate, and ethanol. The ability of the four fractions and EEP to inhibit Streptococcus mutans and Streptococcus sobrinus growth and adherence to a glass surface was examined. The effect on GTFs B and C activity was also determined. For the caries study, 60 Wistar rats infected with Streptococcus sobrinus were treated topically twice daily as follows: (1) EEP type-3, (2) H-fr type-3, (3) EEP type-12, (4) H-fr type-12, and (5) control. In general, the H-fr from both types of propolis showed the highest antibacterial activity and GTFs inhibition. Furthermore, the EEP and H-fr type-3 and -12 were equally effective in reducing dental caries in rats. The data suggest that the putative cariostatic compounds of propolis type-3 and -12 are mostly non-polar; and H-fr should be the fraction of choice for identifying further potentially novel anti-caries agents.

Animals↗
PubMed2006

RT-PCR for detection of all seven genotypes of Lyssavirus genus.

The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

Animals↗
PubMed2006

Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus.

A one step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Plum pox virus (PPV). The six primers required for accelerated RT-LAMP were designed using a conserved region in the C-terminus of the coat protein coding region of PPV. RT-LAMP was used to detect isolates of five strains of PPV including the strains D, M, EA, C, and W. The virus was detected reliably in both infected herbaceous and woody hosts. RT-LAMP was compared to real-time RT-PCR with SYBR Green I and melting curve analysis, using serial dilutions of total RNA extracts. Similar sensitivities were observed, except that real-time RT-PCR was more consistent at lower template concentrations. The purity of the FIP and BIP primers affected the efficiency of the reaction, and incubation time and template concentration affected the ladder-like pattern observed after agarose gel electrophoresis. Although PPV could be detected after 30min of incubation at 63 degrees C, a longer incubation time was required for lower concentrations of the target. RT-LAMP is a very sensitive, low cost diagnostic tool that should be of value in more accurate determination of the distribution of PPV. This should assist in preventing further spread of this devastating virus.

Base Sequence↗
PubMed2006

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.

Animals↗
PubMed2005

Prolactin in the brushtail possum (Trichosurus vulpecula): development of homologous radioimmunoassay using recombinant possum prolactin.

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.

Animals↗
PubMed1997

Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.

Amino Acid Sequence↗
PubMed2001

C-type natriuretic peptide system in rabbit oviduct.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is known to be distributed mainly in brain and vascular endothelium and is considered to act as a local regulator in many tissues. The purpose of this study was to determine the presence of CNP system and its biological function in rabbit oviduct. The serial dilution curve of tissue extracts was parallel to the standard curve of CNP((1-22)) and a major peak of molecular profile of tissue extracts by HPLC was CNP((1-53)). mRNA of CNP which was the same size as positive control was also detected by Southern blot analysis. CNP increased the production of 3',5'-cyclic guanosine monophosphate (cGMP) in the purified membrane of oviduct, which was more in membranes derived from the isthmic portion than in the ampullar portion. The presence of mRNAs of natriuretic peptide receptor-A (NPR-A) and NPR-B was demonstrated by RT-PCR. Synthetic CNP((1-22)) inhibited both frequency and amplitude of basal motility of oviduct in a dose-dependent manner. The inhibitory effect of CNP on the basal motility was more potent in the isthmic portion than in the ampullar portion. These results demonstrate the presence of CNP system in the oviduct and regional differences in motility inhibition by CNP between isthmic and ampullar portions. Therefore, these findings suggest the possible existence of a CNP system that may exert a local regulator of basal motility, either alone or in concert with other hormones.

Animals↗
PubMed2001

C-type natriuretic peptide system in rabbit colon.

C-type natriuretic peptide (CNP) is mainly distributed in the brain and vascular endothelium and is considered to act as a local regulator in many tissues. The present study was aimed to determine the presence of CNP system and its biological function in rabbit colon. The serial dilution curves of tissue extracts were parallel to the standard curve of CNP-22. With gel permeation chromatography and reverse-phase HPLC, the major immunoreactive peak of CNP was observed at the same elution time corresponding to the synthetic CNP-53. The concentration of CNP in the mucosal layer of colon was 212.49 +/- 30.44 pg/g tissue wet weight (n = 7), which was significantly higher than that in the muscular layer. The presence of CNP mRNA was also detected by RT-PCR and Southern blot analysis. Production of cGMP by the activation of particulate guanylyl cyclase stimulated by BNP and CNP was higher in membranes obtained from the muscular layer than from mucosal layer. More cGMP was produced by CNP than by ANP. Both natriuretic peptide receptor-A and -B mRNAs were detected by RT-PCR and specific binding sites to 125I-[Tyr(0)]-CNP-22 were mainly localized to the muscular layer. Synthetic CNP inhibited basal tension, frequency and amplitude of basal motility of taenia coli of the right colon. This study showing the presence of CNP system and its biological function in colon suggests that endogenous CNP synthesized in the mucosal layer may have a paracrine function as a local regulator of colonic motility.

Animals↗