The presence of two distinct prolactin receptors in seabream with different tissue distribution patterns, signal transduction pathways and regulation of gene expression by steroid hormones.

Two prolactin receptors (PRLRs) encoded by two different genes were identified in the fugu and zebrafish genomes but not in the genomes of other vertebrates. Subsequently, two cDNA sequences corresponding to two PRLRs were identified in black seabream and Nile tilapia. Phylogenetic analysis of PRLR sequences in various vertebrates indicated that the coexistence of two PRLRs in a single species is a unique phenomenon in teleosts. Both PRLRs in teleosts (the classical one named as PRLR1, the newly identified one as PRLR2) resemble the long-form mammalian PRLRs. However, despite their overall structural similarities, the two PRLR subtypes in fish share very low amino acid similarities (about 30%), mainly due to differences in the intracellular domain. In particular, the Box 2 region and some intracellular tyrosine residues are missing in PRLR2. Tissue distribution study by real-time PCR in black seabream (sb) revealed that both receptors (sbPRLR1 and sbPRLR2) are widely expressed in different tissues. In gill, the expression level of sbPRLR2 is much higher than that of sbPRLR1. In the intestine, the expression of sbPRLR1 is higher than that of sbPRLR2. The expression levels of both receptors are relatively low in most other tissues, with sbPRLR1 generally higher than sbPRLR2. The sbPRLR1 and sbPRLR2 were functionally expressed in cultured human embryonic kidney 293 cells. Both receptors can activate the beta-casein and c-fos promoters; however, only sbPRLR1 but not sbPRLR2 can activate the Spi promoter upon receptor stimulation in a ligand-specific manner. These results indicate that both receptors share some common functions but are distinctly different from each other in mobilizing post-receptor events. When challenged with different steroid hormones, the two PRLRs exhibited very different gene expression patterns in the seabream kidney. The sbPRLR1 expression was up-regulated by estradiol and cortisol, whereas testosterone had no significant effect. For sbPRLR2, its expression was down-regulated by estradiol and testosterone, while cortisol exerted no significant effect. The 5'-flanking regions of the sbPRLR1 and sbPRLR2 genes were cloned and the promoter activities were studied in transfected GAKS cells in the absence or presence of different steroid hormones. The results of the promoter studies were in general agreement with the in vivo hormonal regulation of gene expression results. The sbPRLR1 gene promoter activity was activated by estradiol and cortisol, but not by testosterone. In contrast, the sbPRLR2 gene promoter activity was inhibited by estradiol, cortisol, and testosterone.

Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11.

Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.

Quantification and characterization of aortic cholesterol in rabbits fed a high-cholesterol diet.

Determination of fat percentage of aortic intimal area stained by Sudan III is useful as an index of atherosclerosis in the rabbit animal model. However, the determination of sudanophilic area of the thoracic aorta is two-dimensional and does not measure the third dimension of depth. The objective of the present study was to quantify and characterize aortic lipids using the gas-liquid chromatographic (GLC) technique and to determine whether elevated measurements of total cholesterol and cholesteryl esters was correlated with increased measurements of sudanophilic area staining of the thoracic aorta in rabbits given either a normal chow or a 1% cholesterol diet. The GLC results showed that there was a mean accumulation of 10.9 mg of cholesterol per gram of aortic tissue in the rabbits given a cholesterol diet (mean sudanophilic area of 23.8%). In contrast, rabbits on a normal chow diet had only a deposition of 0.58 mg of cholesterol per gram of the aortic tissue diet (mean sudanophilic area of 1.4%). The present study suggests that quantification of the aortic lipids can be performed by using GLC techniques and that it could be used as an alternative to the measurement of sudanophilic area when assessing the severity of atherosclerosis.

Difference in absorption of the two structurally similar flavonoid glycosides, hyperoside and isoquercitrin, in rats.

The present study was to investigate oral absorption of the two similar flavonoid glycosides, isoquercitrin (IQ, quercetin-3-O-glucoside) and hyperoside (HP, quercetin-3-O-galactoside) in rats. Two groups of male SD rats received an oral dose of either IQ (4.5 mg/kg) or HP (6.0 mg/kg). Blood samples were collected via jugular vein at time intervals after drug administration and the plasma concentrations of the studied compounds were analyzed by HPLC. The stability of IQ and HP in the GI tract was also measured by incubation with various GI contents from rats. The results showed that unchanged IQ was barely detectable whereas the glucuronidated quercetin (the aglycone of IQ) was found to be the major form in plasma after oral administration of IQ. In contrast, HP could not be detected in plasma neither as unchanged form nor its aglycone or conjugated aglycone form. Additional in vitro stability studies demonstrated that HP is more stable than IQ in the GI tract. This suggests that IQ could be hydrolyzed easier than HP to its aglycone in GI tract before being absorbed. In conclusion, IQ, as a flavonoid glucoside, could be rapidly absorbed and transformed into glucuronidated quercetin and such absorption might be related to the hydrolysis of the type of sugar moieties attached to its aglycone molecule.

Comparison of the pharmacokinetics of hawthorn phenolics in extract versus individual pure compound.

The pharmacokinetics of an active herbal substance may be different when administered in an extract form as compared to that when administered as a pure compound. This study investigated the pharmacokinetics of 4 active compounds of hawthorn fruits--namely, (-)-epicatechin, chlorogenic acid, hyperoside, and isoquercitrin--following administration of an extract formulation (as hawthorn phenolic extract, which contained the active compounds) or equivalent doses of individual pure compound in male Sprague-Dawley rats (n = 5 per group). The hawthorn phenolic extract or pure compounds were administered both orally and intravenously. Following administration, multiple plasma samples were obtained, and the plasma concentrations were determined by high-performance liquid chromatography. After the intravenous injection of hawthorn phenolic extract, higher plasma drug concentration, larger area under the plasma concentration-time curve from 0 to infinity, longer terminal elimination half-life, smaller apparent volume of distribution, lower total body clearance, and higher urinary excretion of each compound were obtained when compared to that after the pure compound. Following the oral administration of either hawthorn phenolic extract or pure compound, only epicatechin was absorbed, and their pharmacokinetics were generally not significantly different between these 2 formulations. The differences in the pharmacokinetics of the 2 formulations following intravenous but not oral administration may be attributable to the existence of other co-occurring components in the hawthorn phonolic extract (which may be present in the body after intravenous but not oral administration). The results showed that an herbal extract formulation, when administered intravenously, could potentially alter the pharmacokinetics of its active ingredients.

Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms.

GH feedback on its own secretion at the pituitary level has been previously reported, but the mechanisms involved have not been elucidated. Here we examined the autocrine/paracrine effects of GH on GH synthesis using grass carp pituitary cells as a cell model. GH receptors were identified in carp somatotrophs, and their activation by exogenous GH increased steady-state GH mRNA levels and GH production. Removal of endogenous GH by immunoneutralization using GH antiserum inhibited basal as well as stimulated GH mRNA expression induced by GH-releasing factors in fish, including GnRH, apomorphine, and pituitary adenylate cyclase-activating polypeptide-38. Cytosolic mature GH mRNA levels were elevated by GH treatment and reduced by GH antiserum, whereas nuclear GH primary transcripts were almost undetectable after GH immunoneutralization. Inhibition of Janus kinase-2 (JAK2), phosphoinositide 3-kinase, and MAPK also abolished GH-induced steady-state GH mRNA expression. GH immunoneutralization in pituitary cells pretreated with actinomycin D induced a marked decrease in the half-life of GH mRNA, indicating that the clearance of GH transcripts could be enhanced by removing endogenous GH. These results provide evidence that GH can serve as a novel intrapituitary autocrine/paracrine factor maintaining GH gene expression in somatotrophs, and this action is mediated by JAK2/MAPK and JAK2/phosphoinositide 3-kinase cascades coupled to GH receptors.

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system.

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.

Hawthorn fruit is hypolipidemic in rabbits fed a high cholesterol diet.

The present study examined the hypolipidemic activity of hawthorn fruit. New Zealand white rabbits were fed one of three diets, a reference diet with no cholesterol added (NC), a high cholesterol diet (1 g/100 g, HC) and a HC diet supplemented with 2 g/100 g hawthorn fruit powder (HC-H). After 12 wk, serum total cholesterol (TC) and triacylglycerols (TG) were 23.4 and 22.2% lower, respectively, in the hawthorn fruit group compared with the HC rabbits (P < 0.05). Hawthorn supplementation led to 50.6% less cholesterol accumulation in aorta (P < 0.05) and 23-95% greater excretion of neutral and acidic sterols (P < 0.05). Supplementation of hawthorn fruit did not affect the activities of hepatic 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA-R) or cholesterol 7alpha-hydroxylase (CH) but it suppressed the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT, P < 0.05). The results suggest that the mechanism by which hawthorn fruit decreases serum cholesterol involves, at least in part, the inhibition of cholesterol absorption mediated by down-regulation of intestinal ACAT activity.