Prolactin-releasing peptide is essential to maintain the prolactin level and osmotic balance in freshwater teleost fish

We administered prolactin-releasing peptide (PrRP) or anti-PrRP antiserum to goldfish in fresh water and analyzed their effects on prolactin and osmoregulatory mechanisms. The pituitary mRNA level of prolactin increased by PrRP but decreased by anti-PrRP. The rate of water inflow in the gills decreased by PrRP and increased by anti-PrRP, showing that PrRP restricts branchial water permeability, as also restricted by prolactin. PrRP also expanded the mucous cell layers on the scales, which may restrict efficiently water inflow by the mucous system. Eventually, the plasma osmotic pressure decreased by anti-PrRP. We conclude that PrRP is essential to maintain prolactin levels and osmotic balance in fresh water.     

Comparative anatomical study of internal brooding in three anascan bryozoans (Cheilostomata) and its taxonomic and evolutionary implications

The anatomical structure of internal sacs for embryonic incubation was studied using SEM and light microscopy in three cheilostome bryozoans-Nematoflustra flagellata (Waters,1904), Gontarella sp., and Biflustra perfragilis MacGillivray, 1881. In all these species the brood sac is located in the distal half of the maternal (egg-producing) autozooid, being a conspicuous invagination of the body wall. It consists of the main chamber and a passage (neck) to the outside that opens independently of the introvert. There are several groups of muscles attached to the thin walls of the brood sac and possibly expanding it during oviposition and larval release. Polypide recycling begins after oviposition in Gontarella sp., and the new polypide bud is formed by the beginning of incubation. Similarly, polypides in brooding zooids degenerate in N. flagellata and, sometimes, in B. perfragilis. In the evolution of brood chambers in the Cheilostomata, such internal sacs for embryonic incubation are considered a final step, being the result of immersion of the brooding cavity into the maternal zooid and reduction of the protecting fold (ooecium). Possible reasons for this transformation are discussed, and the hypothesis of Santagata and Banta (Santagata and Banta1996) that internal brooding evolved prior to incubation in ovicells is rejected.     

Experimental verification of the crucial roles of Glu73 in the catalytic activity and structural stability of goose type lysozyme

The roles of Glu(73), which has been proposed to be a catalytic residue of goose type (G-type) lysozyme based on X-ray structural studies, were investigated by means of its replacement with Gln, Asp, and Ala using ostrich egg-white lysozyme (OEL) as a model. No remarkable differences in secondary structure or substrate binding ability were observed between the wild type and Glu(73)-mutated proteins, as evaluated by circular dichroism (CD) spectroscopy and chitin-coated celite chromatography. Substitution of Glu(73) with Gln or Ala abolished the enzymatic activity toward both the bacterial cell substrate and N-acetylglucosamine pentamer, (GlcNAc)(5), while substitution with Asp did not abolish but drastically reduced the activity of OEL. These results demonstrate that the carboxyl group of Glu(73) is directly involved in the catalytic action of G-type lysozyme. Furthermore, the stabilities of all three mutants, which were determined from the thermal and guanidine hydrochloride (GdnHCl) unfolding curves, respectively, were significantly decreased relative to those of the wild type. The results obtained clearly indicate the crucially important roles of Glu(73) in the structural stability as well as in the catalytic activity of G-type lysozyme.     

Expression and polysome association of YB-1 in various tissues at different stages in the lifespan of mice

Tissue-specific translational regulation is important for gene expression. YB-1 binds to mRNAs to form mRNPs and affects translation. In this study we investigated expression and polysome association of YB-1 in various tissues at different stages in the lifespan of mice. YB-1 levels decreased markedly with growth in brain, heart and muscle, but increased in the spleen. In lung, kidney and testis, the levels of YB-1 diminished with aging. In liver, no significant change in the level of YB-1 was observed throughout life. We further showed that the distribution pattern of YB-1 on a sucrose gradient differed according to tissue. Moreover, the distribution pattern of YB-1 changed drastically with growth in the liver. In 5-day-old liver, YB-1 was distributed almost exclusively in nonpolysomal fractions, whereas in 4-week-old liver, it was associated with heavy-sedimenting polysomes, as was the case in 5-day-old brain. Immunohistochemical analysis revealed that YB-1 is mainly a cytoplasmic protein in these tissues. Our results indicate that the expression and polysome association of YB-1 are regulated with growth or aging in a tissue-specific manner, presumably to control gene expression at the translational level in each tissue.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Are Additional Trace Elements Necessary in Total Parenteral Nutrition for Patients with Esophageal Cancer Receiving Cisplatin-Based Chemotherapy?

It is known that cisplatin induces the excretion of zinc from the urine and thereby reduces its serum concentration. However, the fluctuation of these trace elements during or after cisplatin-based chemotherapy has not been evaluated. To answer this question, we performed a clinical study in esophageal cancer patients undergoing cisplatin-based chemotherapy. Eighteen patients with esophageal cancer who were not able to swallow food or water orally due to complete stenosis of the esophagus were evaluated. The patients were divided into a control group [total parenteral nutrition (TPN) alone for 28?days, ten cases] and an intervention group (TPN with additional trace elements for 28?days, eight cases). The serum concentrations of zinc, iron, copper, manganese, triiodothyronin (T3), and thyroxin (T4), as alternative indicators of iodine, were measured on days?0, 14, and 28 of treatment, and statistically analyzed on day?28. In the control group, the serum concentration of copper was significantly decreased from 135.4 (day?0) to 122.1???g/ml (day?14), and finally to 110.6???g/ml (day?28, p?=?0.015). The concentration of manganese was also significantly decreased from 1.34 (day?0) to 1.17???g/ml (day?14) and finally to 1.20 (day?28, p?=?0.049). The levels of zinc, iron, T3, and T4 were not significantly changed. In the intervention group, the supplementation with trace elements successfully prevented these decreases in their concentrations. TPN with supplementary trace elements is preferable and recommended for patients who are undergoing chemotherapy in order to maintain the patients?? nutrient homeostasis.