Neurochemical studies on imprinting behavior in chick and duckling

The effect of lesiens in the parts of medial hyperstriatum ventrale (MHV) or lateral neostriatum (LN) on impriting behavior was examined. Bilateral lesions in MHV severely impaired imprinting behavior in ducklings, but lesions in LN did not impair such behavior. On the other hand, the birds lesioned in MHV still had brightness discriminative learning ability with perfect performance of visuomotor coordination. The neurotransmitter candidates controlling imprinting behavior in MHV were investigated using blockers of neurotransmission. 6-Hydroxydopamine (6-OHDA) and haloperidole injected into MHV did not have specific effect on imprinting behavior although catecholamine contents in MHV was clearly decreased and locomotor activity was strongly suppressed. Atropine injection into MHV caused significant impairment of following behavior, but locomotor activity was not affected. The injection of atropine into other region did not have any effect. -Bungarotoxin showed no effect on either imprinting or locomotor activity. Kainic acid injection into MHV caused the decrease of glutamate content in the MHV region and it reached maximum at 24 hours after injection. However, imprinting behavior was already impaired at 3 hours after injection, but at 24 hours it had been recovered. A same effect was observed by glutamate injection. The effect of protein synthesis inhibitor on the aquisition process of imprinting was investigated. The aquisition of imprinting was significantly impaired by cycloheximide (CHX) injection into MHV within 2 hours after the first exposure to the imprinting stimulus. When CHX was given 24 hours after, imprinting behavior was not affected.     

Geographic and temporal distributions of four genotypes found in Erysiphe gracilis var. gracilis,a powdery mildew of evergreen oaks (Erysiphales)

A survey of 402 samples of Erysiphe gracilis var. gracilis on evergreen oaks collected from a wide area of western Japan showed that they were divided into four distinct genotypes each forming a separate clade with high bootstrap support, which were referred to as E. hiratae (genotype I), E. uncinuloides (genotype II), E. gracilis s. str. (genotype III), and E. pseudogracilis (genotype IV) in a separate taxonomic treatment. However, there are no clear differences in geographic distributions among these four genotypes. Quercus myrsinifolia was only infected by genotype II and Q. salicina only by genotype IV, whereas Q. glauca was infected by all four. These results strongly suggest an association between host species and speciation of these genotypes. A further 312 samples of Q. glauca with E. gracilis s. lat. colonies were collected from four locations in the Mie University campus to investigate frequency of genotypes I and II every month from May 2015 to January 2016. No temporal isolation was found in genotype frequencies. These genotypes frequently co-existed on a single leaf surface, especially at the locations disturbed by human activities. Two oak powdery mildews, E. gracilis s. lat. and Cystotheca wrightii, produced conidia only one month a year and their life cycle differed from most other powdery mildew species. This suggests that these oak mildews developed their unique life cycles to synchronize with the life cycle of evergreen oaks.     

Russula velenovskyi new to Japan,with phylogenetic implications of Russula species between Japanese subalpine forests and Northern Europe

To compare morphological characters and phylogenetic placement between Japanese and European Russula, 32 specimens of 12 species were collected from Japanese subalpine forests and Northern Europe. Several sequences of nrDNA ITS region (ITS) of these Russula species were obtained. High homological similarities were shown between ITS sequences of several Russula samples collected from Japanese subalpine forests, Europe and North America. These facts show distribution of the same Russula species among these areas. From morphological observations and phylogenetic analyses, two same Russula species, R. velenovskyi, and R. decolorans are found in Japan, Europe and North America. Of these, R. velenovskyi collected from Mt. Fuji, Mt. Nyukasa and Mt. Tateshina in mountainous area of central Honshu is reported as a new Japanese record.     

Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.     

Molecular markers for X‐ray‐insensitive differentiated cells in the Inner and outer regions of the mesenchymal space in planarian Dugesia japonica

Planarian's strong regenerative ability is dependent on stem cells (called neoblasts) that are X‐ray‐sensitive and proliferative stem cells. In addition to neoblasts, another type of X‐ray‐sensitive cells was newly identified by recent research. Thus, planarian's X‐ray‐sensitive cells can be divided into at least two populations, Type 1 and Type 2, the latter corresponding to planarian's classically defined “neoblasts”. Here, we show that Type 1 cells were distributed in the outer region (OR) immediately underneath the muscle layer at all axial levels from head to tail, while the Type 2 cells were distributed in a more internal region (IR) of the mesenchymal space at the axial levels from neck to tail. To elucidate the biological significance of these two regions, we searched for genes expressed in differentiated cells that were locate close to these X‐ray‐sensitive cell populations in the mesenchymal space, and identified six genes mainly expressed in the OR or IR, named OR1, OR2, OR3, IR1, IR2 and IR3. The predicted amino acid sequences of these genes suggested that differentiated cells expressing OR1, OR3, IR1, or IR2 provide Type 1 and Type 2 cells with specific extracellular matrix (ECM) environments.     

Cytoflurometric nuclear DNA-determinations in infant,adolescent, adult and aging human hearts

Summary The progress of polyploidization in the human heart muscle cell was investigated by cytofluorometry, involving selective measurements of heart muscle cell nuclei. Thirty-two tissue samples, taken from the free wall of the left ventricle of each autopsied heart, were fixed in Carnoy's fluid. From thick (100–150 m) paraffin sections, isolated cells for smears were obtained by enzyme digestion and ultrasonic treatment. The smears were stained with azocarmin G to eliminate background fluorescence and subsequently stained by an acriflavine-Feulgen reaction. Cytofluorometric DNA-determinations were carried out selectively on heart muscle cell nuclei, using the muscle striations revealed by azocarmin G-fluorescence as specific markers. The dynamic process of polyploidization in normal hearts could be divided into four stages. In the first stage (under 1 year of age), almost all heart muscle cell nuclei (94.3±1.8%) were diploid. In the second stage (1 to 9 years of age), the number of tetraploid nuclei increased (13.6±7.1%). In the third stage (9 to 22 years of age), octaploid nuclei first appeared and the number of tetraploid nuclei increased (26.7±3.9%). The DNA pattern in the fourth stage (22 to 75 years of age) was relatively constant, with a ratio of diploid (62.4±8.7%), tetraploid (31.4±6.7%) and octaploid (5.8±3.9%) nuclei. From these results it was concluded that physiological polyploidization progresses in proportion to the increase of heart weight. The frequency of polyploid nuclei in human heart was not so high as resported by previous investigators.     

Cerebellar Hypoplasia in the Gunn Rat with Hereditary Hyperbilirubinemia: Immunohistochemical and Neurochemical Studies

Immunohistochemical reactions were conducted, using the antibodies against GFA and S-100 proteins on sections of cerebellum from the homozygous (jj) and the heterozygous (Jj) Gunn rats. Hypertrophy of the fibrous astrocytes was observed but hyperplasia of the glial cells was not. Although the molecular layer was very thin, the Bergmann fibre appeared normal. Among the free amino acids in the cerebellum from the jj rat, glutamate concentration decreased to two-thirds of the control level. The protein profile of the cerebellum from the jj rat obtained by SDS-polyacrylamide gel electrophoresis revealed that the amount of P400 protein that is characteristic of Purkinje cells decreased considerably and there were also some changes of the other unidentified proteins. By two-dimensional electrophoresis, it was observed that in the supernatant from the jj rat cerebellum one protein spot diminished and in the particulate fraction from the jj rat one spot was enormously increased. The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the cerebellum from the jj rat did not differ significantly from that of the control; however, activities of choline acetyltransferase and acetylcholinesterase of the jj rat were about twice as high as those of the control. 2-Deoxyglucose incorporation was maximum in the granular layer from both the jj and the Jj rat cerebella. However, the incorporation in the jj cerebellum was not higher than in the Jj control and even lower in some parts of the jj cerebellum than in the control Jj cerebellum.     

Effect of food restriction on serotonin metabolism in rat brain

The brain concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased in rats maintained on restricted volume of low-protein or normal-protein diet, whereas these two agents decreased in rats fed low-protein diet ad libitum. In these two food-restricted groups brain 5-HT and 5-HIAA concentrations were not correlated with brain tryptophan hydroxylase activity, but the concentrations correlated closely with cerebral tryptophan concentrations. The cerebral tryptophan concentration in the two food-restricted groups was not consistent with the total or free tryptophan concentration in plasma. In these restricted rats cerebral tryptophan concentration was elevated, and, unlike the plasma tryptophan, it showed no diurnal variation. These results suggested that tryptophan uptake into the brain from plasma was enhanced by limiting food volume intake. Tryptophan uptake was increased by glucagon injection without changing the plasma tryptophan level, but injection of hydrocortisone or insulin had little or no effect on tryptophan concentration in either the plasma or brain.d-Glucose injection elevated plasma tryptophan concentration but decreased brain tryptophan concentration.