Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).     

Genetic population structure of the fluvial eight-barbel loach <Emphasis Type="Italic">Lefua</Emphasis> sp. 1 in the three river systems in central Honshu,Japan, revealed by microsatellite DNA markers

The fluvial eight-barbel loach Lefua sp. 1 is an undescribed species distributed from the Kinki to Chugoku districts, Honshu, and also on Shikoku Island, Japan. Genetic relationships among local populations are unclear and management units remain undetermined. To aid conservation, we determined genetic population structures from microsatellite loci for 20 populations from three river systems on Honshu. The genetic diversity within populations is relatively low; the majority has experienced genetic bottlenecks. Statistical analysis revealed significant divergence among river systems suggesting that each should be recognized as a management unit. Any conservation program should consider the populations’ genetic uniqueness.     

Blood brain barrier permeability of (−)-epigallocatechin gallate,its proliferation-enhancing activity of human neuroblastoma SH-SY5Y cells,and its preventive effect on age-related cognitive dysfunction in mice

BackgroundThe consumption of green tea catechins (GTCs) suppresses age-related cognitive dysfunction in mice. GTCs are composed of several catechins, of which epigallocatechin gallate (EGCG) is the most abundant, followed by epigallocatechin (EGC). Orally ingested EGCG is hydrolyzed by intestinal biota to EGC and gallic acid (GA). To understand the mechanism of action of GTCs on the brain, their permeability of the blood brain barrier (BBB) as well as their effects on cognitive function in mice and on nerve cell proliferation in vitro were examined.MethodsThe BBB permeability of EGCG, EGC and GA was examined using a BBB model kit. SAMP10, a mouse model of brain senescence, was used to test cognitive function in vivo. Human neuroblastoma SH-SY5Y cells were used to test nerve cell proliferation and differentiation.ResultsThe in vitro BBB permeability (%, in 30 min) of EGCG, EGC and GA was 2.8±0.1, 3.4±0.3 and 6.5±0.6, respectively. The permeability of EGCG into the BBB indicates that EGCG reached the brain parenchyma even at a very low concentration. The learning ability of SAMP10 mice that ingested EGCG (20 mg/kg) was significantly higher than of mice that ingested EGC or GA. However, combined ingestion of EGC and GA showed a significant improvement comparable to EGCG. SH-SY5Y cell growth was significantly enhanced by 0.05 µM EGCG, but this effect was reduced at higher concentrations. The effect of EGC and GA was lower than that of EGCG at 0.05 µM. Co-administration of EGC and GA increased neurite length more than EGC or GA alone.ConclusionCognitive dysfunction in mice is suppressed after ingesting GTCs when a low concentration of EGCG is incorporated into the brain parenchyma via the BBB. Nerve cell proliferation/differentiation was enhanced by a low concentration of EGCG. Furthermore, the additive effect of EGC and GA suggests that EGCG sustains a preventive effect after the hydrolysis to EGC and GA.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

Changes in the Proliferative Activity of Epidermal Melanocytes in Serum‐Free Primary Culture During the Development of Ultraviolet Radiation B‐Induced Pigmented Spots in Hairless Mice

Long‐term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR‐1 X HR/De)F₁. To clarify the cellular mechanism for the development of these UVB‐induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation. Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum‐free medium supplemented with dibutyryl adenosine 3′:5′‐cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation). Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small‐sized pigmented spots) and 37 (the stage of development of medium‐sized pigmented spots) weeks after the cessation of 8‐week UVB exposure. At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed. With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes. These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB‐irradiated skin increases with the development of pigmented spots.     

Alternate muscle activity observed between knee extensor synergists during low-level sustained contractions.

To determine quantitatively the features of alternate muscle activity between knee extensor synergists during low-level prolonged contraction, a surface electromyogram (EMG) was recorded from the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) in 11 subjects during isometric knee extension exercise at 2.5% of maximal voluntary contraction (MVC) for 60 min (experiment 1). Furthermore, to examine the relation between alternate muscle activity and contraction levels, six of the subjects also performed sustained knee extension at 5.0, 7.5, and 10.0% of MVC (experiment 2). Alternate muscle activity among the three muscles was assessed by quantitative analysis on the basis of the rate of integrated EMG sequences. In experiment 1, the number of alternations was significantly higher between RF and either VL or VM than between VL and VM. Moreover, the frequency of alternate muscle activity increased with time. In experiment 2, alternating muscle activity was found during contractions at 2.5 and 5.0% of MVC, although not at 7.5 and 10.0% of MVC, and the number of alternations was higher at 2.5 than at 5.0% of MVC. Thus the findings of the present study demonstrated that alternate muscle activity in the quadriceps muscle 1) appears only between biarticular RF muscle and monoarticular vasti muscles (VL and VM), and its frequency of alternations progressively increases with time, and 2) emerges under sustained contraction with force production levels < or =5.0% of MVC.     

The 1.6-A crystal structure of the copper(II)-bound bleomycin complexed with the bleomycin-binding protein from bleomycin-producing Streptomyces verticillus.

Bleomycin (Bm) in the culture broth of Streptomyces verticillus is complexed with Cu(2+) (Cu(II)). In the present study, we determined the x-ray crystal structures of the Cu(II)-bound and the metal-free types of Bm at a high resolution of 1.6 and 1.8 A, respectively, which are complexed with a Bm resistance determinant from Bm-producing S. verticillus, designated BLMA. In the current model of Cu(II).Bm complexed with BLMA, two Cu(II).Bm molecules bind to the BLMA dimer. The electron density map shows that the copper ion is clearly defined in the metal-binding domain of the Bm molecule. The metal ion is penta-coordinated by a tetragonal monopyramidal cage of nitrogens and binds to the primary amine of the beta-aminoalanine moiety of Bm. The binding experiment between Bm and BLMA showed that each of the two Bm-binding pockets has a different dissociation constant (K(d)(1) and K(d)(2)). The K(d)(1) value of 630 nm for the first Bm binding is larger than the K(d)(2) value of 120 nm, indicating that the first Bm binding gives rise to a cooperative binding of the second Bm to the other pocket.