The role of brain-gut peptide galanin in the regulation of prolactin (PRL) and beta-endorphin (beta-EP) release from anterior pituitary lobe was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells of the rat. Galanin (1 microgram or 3 micrograms/rat) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of PRL resting secretion, but did not alter resting secretion of beta-EP and restraint stress-induced release of PRL and beta-EP. However, galanin (0.05, 0.5 and 1.0 micrograms/5 x 10(5) cells.ml-1) induced highly significant, dose-related increase of beta-EP secretion from dispersed anterior pituitary cells, but failed to alter significantly PRL secretion from the cultured cells. These results indicate that central galanin has a stimulatory role in pituitary PRL resting secretion via the hypothalamus, whereas peripheral galanin stimulates beta-EP secretion only via direct action of this peptide in anterior pituitary cells.
To investigate the effects of ghrelin on delayed gastrointestinal transit in alloxan-induced diabetic mice.A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups: normal mice group and diabetic mice group treated with ghrelin at doses of 0, 20, 50, 100 and 200 mug/kg ip. Gastric emptying (GE), intestinal transit (IT), and colonic transit (CT) were studied in mice after they had a phenol red meal following injection of ghrelin. Based on the most effective ghrelin dosage, atropine was given at 1 mg/kg 15 min before the ghrelin injection for each measurement. The mice in each group were sacrificed 20 min later and their stomachs, intestines, and colons were harvested immediately. The amount of phenol red was measured. Percentages of GE, IT, and CT were calculated.Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice (22.9 +/- 1.4 vs 28.1 +/- 1.3, 33.5 +/- 1.2 vs 43.2 +/- 1.9, 29.5 +/- 1.9 vs 36.3 +/- 1.6, P < 0.05). In the diabetic mice, ghrelin improved both GE and IT, but not CT. The most effective dose of ghrelin was 100 mug/kg and atropine blocked the prokinetic effects of ghrelin on GE and IT.Ghrelin accelerates delayed GE and IT but has no effect on CT in diabetic mice. Ghrelin may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.
Using Light and electron microscopy the fine structure of the thyroid gland and the adrenal gland of finless porpoises from Yangtze River were observed. The results show that the structure of the thyroid gland is similar to that of any other mammals. But the number of the parafollicular cells is large and these cells constitute a large part of some follicles. With respect of the ultra-structure of the thyroid gland, four types of follicular cells are observed. It is thought that those different types of cells may be the expression of the follicular cells under physiologically different states. In addition, microvillus or fold on the apical and the basal surface of the follicular cells are not seen. The structure of the adrenal gland is also rather similar to that of any other mammals and is usually composed of cortex and medulla. The adrenal cortex contains zona glomerulosa, zona fasciculate and zona reticularis. But the zona glomerulosa is sometimes absent. Two types of adrenal cortical cells are observed by electron microscopy. In one type of cells, a large number of the rough endoplasmic reticula are present, while in another type of cells, the smooth endoplasmic reticula and mitochondria are very abundant.
As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.
The effect of cholecystokinin octapeptide (CCK-8) on the release of prolactin (PRL) in male rats were studied in vivo and in vitro. CCK-8 at the concentrations (microgram) of 0.05 and 0.5 was injected into the third cerebral ventricle (3rd, V. I) of conscious rats, outfitted with chronic 3rd. V. and jugular cannulae, a significant increase in resting secretion and restraint stress-induced release of PRL were observed. The effects of CCK-8 at the concentration of 0.05 microgram were stronger than those of 0.5 microgram. To determine if CCK-8 would exert any direct action on anterior pituitary, CCK-8 of 0.05, 0.5, 1.00 microgram were added to the medium of dispersed anterior pituitary cell, and caused dose-dependent increase of PRL secretion. To study a mechanism of intracellular signal transduction in the action of CCK-8, the levels of cAMP and [Ca2+] in the medium were measured. Intracellular Ca2+ concentration of disperse anterior pituitary cell was significantly elevated by CCK-8 (2 x 10(-4) mol/L), but CCK-8 (10(-8)-10(-6) mol/L) did not change intracellular cAMP content. The results indicate that CCK-8 stimulate prolactin release at both sites of hypothalamic and anterior pituitary and the mechanism of stimulating effects of CCK-8 might be mediated by [Ca2+] but not cAMP.
To investigate the effect of nonylphenol (NP) on testosterone secretion of Leydig cells.A primary culture system of Leydig cells was set up, followed by identification of Leydig cells with 3beta-HSD staining. After treatment with different concentrations of NP, testosterone secretion was detected and morphological examination was performed.Following treatment with NP, morphologic changes of Leydig cells were detected, with decreased cell density at high doses of NP. Testosterone increased at lower concentrations of NP while decreased at high concentrations of NP.Lower doses of NP can stimulate the secretion of testosterone, but increased exposure to NP will inhibit testosterone secretion of Leydig cells. Besides, high concentrations of NP can cause death of Leydig cells in vitro.
Premature Sprague-Dawley male rats were treated with testosterone propionate for 15 days. The levels of testosterone and estradiol were detected by radio-immuno assay. The population of lymphocyte and its subsets was determined by flow cytometry. MTT colorimetric assay was used to show the proliferative ability of lymphocytes induced by Con A. ELISA was performed to test the levels of IL-6 and IgG. The testosterone can downregulate serum levels of IL-6, IgG and the ratio of CD4/CD8. On the other hand, it can increase the population of CD8 lymphocytes. Testosterone exerts an inhibiting effect on the immune system. Androgen therapy may be used in some auto-immune diseases.
Abstract Background Navigating the intricacies of discovering unique and functional antibody binders for in vitro diagnostic development comes with several technological challenges, such as limited diversity of antibody repertoires, functional screening challenges, and the absence of suitable in vitro models. A proteomics-driven strategy to antibody discovery is a promising solution to overcome these obstacles by facilitating polyclonal to monoclonal antibody conversion. De novo polyclonal antibody sequencing utilizes mass spectrometry and machine-learning driven bioinformatics to obtain complete antibody sequences from immunoserum or purified protein samples. This approach investigates the entire circulating antibody repertoire at the protein level, thereby broadening the spectrum of potential candidates. It further facilitates functional selection by enabling antibody purification and enrichment techniques. This capability facilitates the identification of antibodies possessing unique functions, crucial for subsequent downstream development. We demonstrate the application and de novo polyclonal antibody sequencing using mass spectrometry and machine learning bioinformatics to enable the next generation of antibody discovery. Methods In this study, we employed a novel mass spectrometry-based approach, REpAb, to de novo sequence polyclonal antibodies directly from the immunized serum of animals or human patients. REpAb integrates mass spectrometry with machine learning-based bioinformatics to de novo sequence and assemble monoclonal antibodies from a polyclonal mixture. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark). Results De novo polyclonal sequencing with REpAb yielded unique antibody binders not found in splenocytes or peripheral blood mononuclear cells (PBMCs). De novo antibody sequencing-discovered antibody sequences from naturally exposed humans showed unrestricted germline usage, compared to restricted germline used in antigen-immunized animals. Additionally, a proteomics-based approach for antibody discovery enables functional antibody selection through standard protein A/G purification, followed by negative and positive selection enrichment strategies to isolate antibody clones specific to the region of interest on the antigen. Conclusions Sequencing antibodies at the protein level directly reflects the circulating antibody repertoires, which expands the antibody diversity from the BCR repertoire. Proteomic-based antibody discovery empowers the functional selection of antibodies via meticulously designed enrichment strategies. This approach allows for the identification of a diverse pool of candidates possessing specific functions, such as anti-idiotypic features.
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