Tetsuo Nasu

Comparison of circadian oscillation of melatonin release in pineal cells of house sparrow, pigeon and Japanese quail, using cell perfusion systems

We compared the circadian oscillation of melatonin release from cultured pineal cells in Japanese quail, pigeon and house sparrow, to determine whether the pineal gland of these species retains the circadian oscillator function in vitro. After dissociated pineal cells have been cultured for 4 days under 12 h: 12 h light-dark (LD) cycle, they were perfused at a flow rate of 0.25 ml/h for 6-7 days under LD or constant darkness (DD). Melatonin release increased during the dark period and low during the light period in all pineal cell cultures. Under DD conditions, the circadian rhythm of melatonin release persisted for up to 3-4 cycles in pigeon and house sparrow pineal cells, but the amplitude of the rhythm decreased gradually. However, in Japanese quail pineal cell culture the circadian oscillation of melatonin release was weak or abolished under DD conditions. These results strengthen the argument that the avian pineal glands role in circadian organization differs between species. The direct demonstration of species-specific differences in the mechanism of the circadian oscillation of melatonin release from pineal cells should provide a useful model for the analysis of the pineals circadian system at the cellular level.

Individual pineal cells in chick possess photoreceptive, circadian clock and melatonin-synthesizing capacities in vitro

Chick pineal cells express a circadian rhythm of melatonin release under light-dark (LD) cycles, with an increase during the dark period and a decrease during the light period, and this rhythm persists under constant darkness (DD). We cultured individual single pineal cells with 15 microl of medium per well in a Terasaki plate and measured melatonin secretion every 12 h under LD, DL and DD. Individual cells secreted more melatonin during the dark period than during the light period under both LD and DL conditions, and those rhythmic secretions persisted under DD. These results suggest that individual pineal cells in chick have photoreceptive, circadian clock and melatonin-synthesizing capacities.

Development of regulation of melatonin release in pineal cells in chick embryo.

Melatonin release in a pineal cell culture from 13- and 14-day-old chick embryos increased during the dark phase and decreased during the light phase of a 12 h light:12 h dark cycle. When the light-dark cycle was reversed, the pattern of melatonin release in the culture also reversed. 8-Bromo cyclic-AMP stimulated melatonin release in both the light and dark phases. However, no rhythm of melatonin release was detected under constant dark (DD) conditions in a cell culture from 14-day-old chick embryos. In 18-day-old chick embryos, the pineal cell culture expressed a circadian rhythm of melatonin release under DD conditions. These results indicate that mechanisms regulating melatonin synthesis in the avian pineal gland are established during embryonic life.

Melatonin release from pineal cells of diurnal and nocturnal birds

Melatonin release from the pineal cells of chicks, pigeons and crows (diurnal birds) in vitro was compared with that from owls (a nocturnal bird). The pineal cells of diurnal birds secreted large amounts of melatonin during the dark period, whereas owl pineal cells released virtually no melatonin over 24 h and did not respond to exogenous stimulant agents. Histological examination revealed that the owl pineal gland is very small and has a poor vascular network. These results suggest that the pineal gland of owls may have degenerated and is not involved in the circadian clock mechanism in this species.

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Abstract Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development.

Clindamycin in the Treatment of Babesia gibsoni Infections in Dogs

This report examines the effectiveness of clindamycin for the treatment of babesiosis in dogs (n=10) experimentally infected with Babesia gibsoni (B. gibsoni). Clindamycin (25 mg/kg body weight, per os, q 12 hours for 14 days) gradually reduced parasitemia levels and induced morphological changes that indicated degeneration of parasites (e.g., segmentation; size reduction; localization in the cell limbic and/or torn state of the nucleus; and swelling, decrease, or disappearance of the cytoplasm) in the majority of dogs. Clindamycin treatment reduced the clinical symptoms characteristic of Babesia infection, including anemia, anorexia, and listlessness. Clindamycin might be useful as a medicine for treatment of B. gibsoni infection.

Effect of glucagon-like peptide-1 and -2 on regulation of food intake, body temperature and locomotor activity in the Japanese quail

To investigate the physiological roles of glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) in avian species, we elucidated the effect of intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of GLP-1 and GLP-2 on food intake, body temperature and gross locomotor activity in adult Japanese quail. Both i.p. and i.c.v. administration of GLP-1 suppressed food intake at 2, 4 and 12h after administration. Moreover, both i.p. and i.c.v. administration of GLP-1 significantly decreased both body temperature and gross locomotor activity 2h after administration. On the other hand, both i.p. and i.c.v. administration of GLP-2 had no effect on food intake, body temperature or gross locomotor activity. These results suggest that GLP-1 may have an important role in the regulation of food intake, body temperature and locomotor activity while GLP-2 may have no apparent effect on feeding regulation in adult Japanese quail.

Effect of microbial transglutaminase on the natural actomyosin cross-linking in chicken and beef.

The objective of this research was to investigate the difference between chicken and beef in the interaction of actomyosin (myosin B) with microbial transglutaminase (MTG). The gel strength of myosin B was improved in both species and was significantly greater in beef than in chicken (P<0.01). The degree of protein viscosity and the ε(γ-glutamyl)lysine (G-L) content were significantly higher in beef than in chicken (P<0.01). Myosin heavy chain (MHC) bands visualized by SDS-PAGE revealed that the same proteins in various meat species vary in their size and structure. Scanning electron microscope images (SEMI) revealed that myosin B in both species was polymerized, and formed multi-projection structures of G-L; surprisingly, more of these structures were found in beef than in chicken. It is possible that the proteins in chicken are folded into a strand shape that tightly encases a considerable number of glutamine and lysine residues, whereas MTG substrate cannot couple glutamine and lysine. This suggests that the reactivity of MTG is dependent on the residual amino acids present on the surface of myosin B in meat. Some protein components (peptides with long reiterated methylene groups attached) joined by disulfide bonds (cysteine) in chicken samples were inhibitory and reduced MTG activity. SEMI also suggested that all MTG-dependent mega-structures of protein molecules generated in chicken and beef may vary greatly in size, configuration and complexity after treatment with MTG. We concluded that the optimal cross-links in myosin B induced by MTG are heterogeneous in chicken and beef.

Impact of transglutaminase on the textural, physicochemical, and structural properties of chicken skeletal, smooth, and cardiac muscles

This study examined the effects of microbial transglutaminase (MTG; 3.1mg/ml) on chicken skeletal, smooth, and cardiac muscles; the meat containing the different muscle types was shaped into sausages and treated at 40°C and/or 78°C for 30min. Although the three muscle types were obtained from the same bird, the effects of MTG addition were not uniform. All the muscle types showed a significant increase in the breaking strength (P<0.01), but skeletal muscle exhibited the maximum increase. All samples showed a decrease in the fluorescence intensity and a significant reduction in the concentration of proteins that were extracted in a high ionic strength solution (P<0.05). Scanning electron microscopy images and histological studies revealed that different muscle types had different physical structures and frameworks after MTG treatment, which is a reflection of the differences in the reaction specificity of MTG with different muscle proteins. Histological studies revealed that the reactions of MTG with meat proteins are both exogenous and endogenous. Cooking loss data suggested that MTG did not have any negative effect on water retention during cooking. MTG appears to be a functional and contributive substance since the results suggest that MTG can function on all muscle types that are mechanically processed for different industrial applications. MTG aggregates muscle proteins in different ways that improve their organoleptic properties such as texture, appearance, and water retention.

Involvement of protein kinase A in the subjective nocturnal rise of melatonin release by chick pineal cells in constant darkness

Nakahara K, Murakami N, Nasu T, Kuroda H, Murakami T. Involvement of protein kinase A in the subjective nocturnal rise of melatonin release by chick pineal cells in constant darkness. J. Pineal Res. 1997; 23:221–229.