Tissue-specific expression of the NOD-like receptor protein 3 in BALB/c mice

Activation of the innate immune system requires recognition of pathogen-associated molecular patterns, such as NOD-like receptors. The NOD-like receptor protein 3 (NLRP3) inflammasome is involved in induction of the pro-inflammatory cytokine, IL-1β, and subsequent inflammatory responses. NLRP3 inflammasome plays important roles in the inflammatory and innate immune responses associated with autoimmune/inflammatory syndrome. However, analysis of the tissue distribution and expression profiles in BALB/c mice is still incomplete. In this study, we investigated the tissue distribution and expression pattern of NLRP3 in BALB/c mice to further elucidate its function in innate immunity in this commonly used laboratory animal model. NLRP3 mRNA expression levels and tissue distribution of the protein were investigated by real-time quantitative PCR and immunohistochemical analyses, respectively. NLRP3 mRNA expression was higher in the kidney and inguinal lymph nodes than in other tissues. Cytoplasmic expression of NLRP3 was detected in the epithelial reticular cells of the spleen and thymus, lymphocytes in the inguinal lymph nodes, cardiac muscle cells, cerebral cortex neurons, alveolar macrophages, renal tubule cells and liver sinusoidal endothelial cells. The results of this study will assist investigators in interpreting site-specific functions and roles of NLRP3 in inflammatory responses.     

Stable isotope variations in otoliths of Pacific halibut (Hippoglossus stenolepis) and indications of the possible 1990 regime shift

Stable oxygen and carbon isotope ratios (δ¹⁸O and δ¹³C) from archived otoliths of Pacific halibut, Hippoglossus stenolepis, were measured to examine the most recent regime shifts in British Columbia and the Gulf of Alaska. δ¹⁸O values of these otoliths ranged from −1.5 to +2.8‰ VPDB, and were consistent with the life stages and migration behavior of halibut. δ¹³C varied from −3.3 to +0.9‰ VPDB, but did not show a transition from the juvenile to the adult stage as does δ¹⁸O. Evaluation of δ¹⁸O and δ¹³C values of mature halibut (ages 8–12) indicated that the 1977 regime shift might have a warming impact on the northeast Pacific fish stocks. In contrast, a possible regime shift around 1990 with a bottom seawater temperature decrease of about 2 °C might have occurred in both the areas. The connection between stable isotope variations in otoliths and the climate regime shifts is thus potentially useful in studying the population dynamics of Pacific halibut, and decadal scale (e.g., the last 20–30 years) ocean environmental changes.     

In vitro synthesis of 20α-reduced and of 11- and 21-oxygenated steroids and their sulfates by testes of the goldfish (Carassius auratus): Testicular synthesis of corticosteroids

Testes from spermiating goldfish were incubated with [³H]17-hydroxyprogesterone. The major products in the unconjugated fraction were identified as androstenedione, androstenetrione, 11-β-hydroxyandrostenedione, 11-ketotestosterone, 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) and 11-deoxycortisol. Testosterone was present predominantly in the glucuronide fraction, but yields were low (1–3%). The major components of the sulfate fraction were 17,20αP and 11-deoxycortisol. The identification of cortisone in low yield (< 2.5010) in both the free and sulfate fractions is the first report of corticosteroid biosynthesis by a teleost testis. The high yields of 17,20αP and 11-deoxycortisol and their sulphates suggests that their possible role in spermiation of the goldfish should be further investigated.     

Preparation and comparison of biological properties of recombinant carp (Cyprinus carpio) growth hormone and its Cys-123 to Ala mutant

Carp growth hormone (cGH) cDNA, in which Cys-123 was mutated to Ala, was prepared, transferred to the expression vector, expressed in Escherichia coli and the mutant was purified to homogeneity. The mutation only slightly improved yield of the monomeric fraction, indicating that Cys-123 is not involved in improper refolding. As compared to cGH, the mutant (cGH-C123A) exhibited lower binding affinity toward homologous liver receptors and lower bioactivity in a 3T3-F442A preadipocyte bioassay despite the fact that both hormones exhibited almost identical cross-reactivity with anti-cGH antibodies. These results, along with those of a structural comparison to hGH, suggest that Cys-123 is located in the hydrophobic core of the hormone, and is most likely affecting the conformation of the binding site. Dimeric forms of the hormone and its mutant were less active than their respective monomers. Homologous binding experiments using a carp liver microsomal fraction revealed a single receptor population with Kd = 0.77 nM and B_max = 241 fmol/mg microsomal protein.

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Résumé Un ADN complémentaire (cDNA) de l'hormone de croissance de carpe (cGH), dans lequel l'acide aminé Cys-123 a été muté en Ala, a été préparé, inséré dans un vecteur d'expression, et exprimé dans Escherichia coli. Le mutant a ensuite été purifié jusqu'à homogénéité. La mutation améliore seulement faiblement la production de la fraction monomérique, indiquant que le Cys-123 n'est pas impliqué dans un repliement erroné. Comparé à la cGH, sa forme mutée (cGH-C123A) montre une plus faible affinité de liaison vis à vis de récepteurs hépatiques homologues, et une plus faible activité biologique dans un test réalisé sur des préadipocytes 3T3-F442A; cela en dépit du fait que les deux hormones présentent des réactions croisées presque identiques avec un anticorps anti-cGH polyclonale. Ces résultats, associés à une comparaison à la structure de l'hGH, suggèrent que le Cys-123 est localisé dans la partie hydrophobique de l'hormone, et affecte, le plus vraisemblablement, la conformation du site de liaison. Les formes dimériques de l'hormone et de sa forme mutée sont moins actives que leurs monomères respectifs. Les études de liaison homologue, réalisées avec des fractions microsomales de foie, révèlent une population unique de récepteurs de Kd = 0,77 nM et de B_max = 241 fmol/mg de proteine microsomale.

     

Control of ion transport by the sperm duct epithelium of brook trout (Salvelinus fontinalis)

The sperm duct epithelium of brook trout (Salvelinus fontinalis), mountedin vitro in Ussing-style epithelial chambers actively absorbs Na⁺ (measured as the short-circuit current, I_sc) and secretes K⁺ (measured using⁸⁶Rb⁺ as tracer). Dibutyryl-cyclic-adenosine monophosphate (db-cAMP) and 3-isobutyl-1-methylxanthine (IMX) produce a rapid, sustained stimulation of both ion transport processes, but the hormone connected to the response is unknown. Purified sockeye salmon CON A2 gonadotropin (GtH) produces a dose-dependent, rapid and sustained rise in Na⁺ uptake and K⁺ secretion. The time course, electrophysiological and transport characteristics are similar to those evoked by IMX. Carbohydrate-poor (chum salmon CON A1) GtH is ineffective. Pretreatment of fish with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) significantly increases milt volume but is without effect on resting or stimulated (IMX + db-cAMP) levels of sperm duct ion transport. This is the first indication of a direct, rapid action of GtH on ion transport by the vertebrate blood-testis barrier. The results suggest direct involvement of GtH in control of later stages of sperm maturation.     

Substrate concentration affects the in vitro metabolism of 17-hydroxyprogesterone by ovaries of the carp,Cyprinus carpio

Carp ovarian tissue was incubated with ³H-17-hydroxyprogesterone in the presence of 0, 0.1, 1, 10, and 100 μg ml⁻¹ unlabeled 17-hydroxyprogesterone. The pattern of metabolites formed showed a marked variation with substrate concentration. Formation of glucuronide and sulphate conjugates was important only at low substrate concentration. At high substrate concentration (10 and 100 μg ml⁻¹) 17,20α-dihydroxy-4-pregnen-3-one was the major metabolite, but at intermediate concentrations polar 7α-hydroxypregnanetetrols predominated. The results support the hypothesis that at low substrate concentrations conjugating, 5α-reducing and 7α-hydroxylating enzymes, of high activity but low capacity, act as scavengers to deactivate any steroids formed during the relatively low pituitary gonadotrophin secretions which are necessary for oocyte development, but that during the prespawning gonadotrophin surge when high levels of substrate are present these enzymes are saturated and 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) becomes the major ovarian steroid. The possible role of 17,20αP during oocyte final maturation requires further examination.     

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17a, 20-dihydroxy-4-pregnen- 17,20-P) and 17,20,21-trihydroxy-4-pregnen-3-one (20-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20-P and 20-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20-P. Serum levels of 17,20-P and 20-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20-P and 20-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20-P and 20-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20-P and 20b-S as MIHs in the bambooleaf wrasse.     

Total and free thyroid hormones in plasma of tropical marine teleost fish

Plasma levels of L-thyroxine (T₄) and 3,5,3-triiodo-L-thyronine (T₃) and the percentage of plasma T₄ and T₃ present in the free (dialyzable) form (%FT₄ and %FT₃) were measured in 16 species (11 families) of tropical marine teleosts from an inshore Barbados reef. Mean plasma T₄ varied from 0.2 ng/ml to 42 ng/ml; mean plasma T₃ varied from < 0.2 ng/ml to 50 ng/ml. The highest T₄ and T₃ levels were recorded in parrot-fish and the lowest levels in filefish. The %oFT₄ and %FT₃ varied from 0.05–3.41%. Estimated levels of plasma free T₄ and free T₃ levels ranged from 0.4–466 pg/ml. The extremely wide inter- and intra-species ranges in levels of free T₄ and T₃ do not support a previous suggestion, based on temperate freshwater salmonid species, that free T₄ and T₃ levels in fish may fall within a relatively range narrow comparable to that of homeothermic vertebrates.