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Objectives: To evaluate the efficacy of tegafur–uracil (UFT), a prodrug of 5-fluorouracil, plus cisplatin and dexamethasone in patients with docetaxel-refractory prostate cancers. Methods: Twenty-five patients with docetaxel-refractory prostate cancer were administered oral UFT plus intravenous cisplatin (UFT-P therapy) and dexamethasone. Treatment responses were assessed monthly via prostate-specific antigen (PSA) level measurements. Treatment-related adverse events and overall survival were also assessed. Results: UFT-P therapy resulted in decreased PSA levels in 14 (56%) patients and increased PSA levels in 11 (44%). In patients with increased PSA levels, 7 (64%) of the 11 patients displayed decreased PSA doubling times. The UFT-P therapy response rate was 84% (21/25 patients). Imaging studies revealed that tumor shrinkage during UFT-P therapy occurred in 1 patient in whom bilateral hydronephrosis caused by lymph node metastasis improved. The median survival time from docetaxel initiation was 36 months. In UFT-P-treated patients, the median PSA progression and overall survival times were 6 and 14 months, respectively. UFT-P treatment-related adverse events were mild diarrhea, general fatigue, and anorexia. Treatment was not discontinued for any of the patients. UFT-P therapy did not cause serious hepatic or renal dysfunction or pancytopenia. Conclusions: UFT-P therapy is a safe and effective treatment for patients with docetaxel-refractory prostate cancer, although large-scale, multicenter, prospective studies are needed to validate these findings.

Malignant bowel obstruction (MBO), an occasional complication in patients with advanced urological cancer, causes gastrointestinal symptoms such as nausea and vomiting leading to suffering which severely impairs quality of life (QOL) . Drug therapy, especially octreotide, a synthetic analog of somatostatin, is reportedly effective in controlling the symptoms of MBO. In the present study, we administered octreotide to urological cancer patients with MBO and evaluated the improvement of subjective symptoms, oral intake, and nasogastric intubation. Fourteen terminally ill urological cancer patients suffering with MBO were included (age range 55-92, 10 male, 4 female). Octreotide was administered at 300µg/day to those patients subcutaneously as a continuous injection. Significant improvements in subjective symptoms were observed in thirteen patients (92.8%), and ten patients (71.4%) were able to resume oral intake. Four patients required nasogastric drainage before the administration of octreotide, but nasogastric intubation was discontinued in all these cases after the use of octreotide. Early initiation of octreotide resulted in better improvement of MBO symptoms, and no adverse event was observed in any of the patients. These results revealed that 300µg/day dose of octreotide is safe and effective for managing gastrointestinal symptoms of terminally ill urological cancer patients with MBO. We also recommend starting the treatment with ocreotide as soon as MBO is diagnosed.

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

Tragulidae is a primitive ungulate family within the order Cetartiodactyla, suborder Ruminantia. Domestic ruminants such as cattle, sheep, and goat have two types of Peyer's patches (PP): jejunal and ileal PP, in which there are morphological and functional differences. In this study, lesser mouse deer (Tragulus javanicus) PP was studied by gross anatomical and histological procedures. At the fetal stage, both types of PP were formed in the small intestine. Ileal PP was observed as a single continuous aggregation of lymphatic follicles extending cranially from the ileo-caecal junction. However, jejunal PP was observed as multiple and discrete accumulations of lymphatic follicles. This study showed that the lesser mouse deer has two types of PP in the small intestine. In addition, the anatomical and histological characteristics of jejunal and ileal PP are quite similar to those of other ruminants' jejunal and ileal PP. Further studies are needed to analyze immune function of both PP in lesser mouse deer in order to determine the evolutionary process of Cetartiodactyla.

This study demonstrated the immunohistochemical localization of cytoskeletal proteins, cytokeratin, desmin, vimentin, α-tubulin and α-smooth muscle actin (α-SMA) in the testes of adult and immature lesser mouse deer (Tragulus javanicus). Desmin was found in the sub-epithelial myoid cells of epididymal ducts. The immunoreactivity for vimentin was shown in the peritubular myoid cells and around the nuclei of the Sertoli cells of which the positions indicated various arrangements. In the Sertoli cells, vimentin filaments between the basement membrane and the nucleus were elongated with the migration of the nucleus toward the upper part. Furthermore, vimentin was detected in the Leydig cells and the sub-epithelial myoid cells of epididymal ducts. Alpha-tubulin was apparently extended to the tip of each Sertoli cell cytoplasm, and α-SMA was found in the peritubular myoid cells and the sub-epithelial myoid cells of epididymal ducts. On the other hand, cytokeratin and desmin were not detected in the Se...

1. Inter-breed morphological comparisons were made among 11 breeds of Japanese native chickens (Gifujidori, Hinaidori, Shokoku, Totenko, Tomaru, Satsumadori, Shamo, Koshamo, Koeyoshi, Chabo and Nagoya), White Leghorn, broiler chickens (Chunky) and red junglefowl collected in the Philippines, based on results of direct measurements and analysis by computer vision techniques of the skull. 2. Analysis of direct measurements identified two groups of chicken: a small type that included the Chabo, Koshamo, red junglefowl, Gifujidori and Shokoku and a large type that included the remaining breeds studied. These groupings were made based on size determined both in the first (PC1) and second principal component (PC2). The greatest length of the cranium and condylobasal length greatly contributed to the morphological differences between these two groups. 3. Analysis by computer vision techniques, however, identified three groups: the Bantam group (which includes red junglefowl), Shokoku group and Shamo group. White Leghorn clustered within the Shokoku group while the broiler chicken belonged to the Shamo group. The region around the junction of the neural cranium and the visceral cranium contributed greatly to the morphological differences among breeds, both in the PC1 and PC2.

Polyethylene glycol (PEG) has been reported to inhibit the development of colonic lesions in carcinogen-treated rats when administered orally. However, the precise mechanism for the chemopreventive activity of PEG remains largely elusive. Based on a characteristic feature of PEG as a 'fusogen', we investigated its potential as a chemotherapeutic agent through the induction of multinucleated cell formation and apoptosis induction in PC-3 prostate cancer cells. When PC-3 cells were treated with 0.5 and 1.0% PEG 1000, multinucleated cells were induced at a frequency of 8.4 and 13%, respectively, 36 h after PEG treatment under high cell density (1 x 10(6) cells in 100 microL PEG solution) in vitro. Although abnormality of cell cycle progression was not evident in PEG-treated PC-3 cells, multinucleated cells substantially disappeared at around 38 h due to apoptosis. In contrast, no apparent growth suppression was observed when PC-3 cells were exposed to up to 1.0% PEG at a much lower cell density, namely under ordinary culture conditions. Furthermore, injection of 0.5% PEG solution in vivo into PC-3 xenografts implanted in BALB/c-nu/nu male mice significantly suppressed tumor growth compared to phosphate-buffered saline injection. Multinucleated TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were observed inside the PEG-injected tumors. PEG was here demonstrated to have anticell proliferation and antitumor effects via induction of apoptosis, possibly by cell fusion. PEG injection therapy could therefore be adopted as an alternative chemotherapeutic strategy for localized prostate cancers, including those that become refractory to androgen-deprivation therapy.

We examined unique erythrocyte pits of the peripheral blood and bone marrow in the lesser mouse deer, Tragulus javanicus, using scanning electron microscope (SEM) and transmission electron microscope (TEM). Under the SEM observation, the pit was observed as a hole on both mature erythrocytes of the peripheral blood and immature erythrocytes of the bone marrow. By the TEM, the mature erythrocytes had a vacuole, which showed complicated shape and occupied considerable space within the cytoplasm. The vacuole was communicated extracellularly by perforation, which corresponded to the hole on the cell surface. In the bone marrow, erythroblast and reticulocytes have a cytoplasmic vacuole. This abnormal feature of the erythrocytes is peculiar to the mouse deer, and not found in other tropical ruminants. Despite the disadvantage of volume loss from the small erythrocytes, the mouse deer were healthy and showed no signs of anaemia.

The Tragulidae are the living relics of the basal ruminant stock. They have a diffuse placenta, with no aggregations of the placental villi into localised placentomes characteristic of all other ruminants. Despite this difference, this ultrastructural and immunocytochemical investigation demonstrates that in Tragulus the trophoblast binucleate cell (BNC) plays the same central role in development and structure as in all other ruminants. It shows an identical development and ultrastructure, produces granules reactive with bovine placental lactogen and pregnancy associated glycoprotein antibodies, and migrates when mature through the trophoblast tight junction to fuse into a mosaic of syncytial plaques from which the granules are released to the mother and which have replaced the uterine epithelium. Unlike the persistent plaques in the sheep and goat placenta, in Tragulus they are transient, dying by apoptosis with the fragments phagocytosed by the trophoblast. This brings the trophoblast into direct endotheliochorial apposition to maternal tissue until BNC migration and fusion replace the dead plaque. This intimate fetomaternal confrontation has not been shown in any other ruminant, and could be a relic of the evolutionary development of the synepitheliochorial from the original basic eutherian endo- or hemo-chorial placenta.

The geographical variations of the skull in the lesser false vampire (Megaderma spasma) were osteometrically compared between the traditionally-accepted subspecies from Philippines, Borneo, Java, North and South Thailand-Peninsular Malaysia. In female the population from Philippines was larger than any other population in skull size, whereas in male, the size tendency was not clearly found. The population of North Thailand was smaller than the four other populations at least in mean value of measurements in both sexes. The canonical discriminant analysis essentially separated the populations in the scattergrams of discriminant scores. However, the plots of Java, North Thailand and South Thailand-Peninsular Malaysia populations were intermingled in male, and Borneo population could not be obviously separated from the Java and South Thailand-Peninsular Malaysia populations in female.

The intracranial carotid rete (or rete mirabile epidurale) is a unique blood vascular system supplying the brain of artiodactyls, which have either an involuted or no internal carotid artery. Although the lesser and greater mouse deer (Tragulus javanicus and T. napu, respectively) are ruminants, the rete mirabile epidurale is absent. In these animals, as in non-artiodactyls, such as canines, equines and humans, the complete internal carotid artery supplies the brain. It is currently uncertain whether the absence of the rete is confined to mouse deer among ruminants. The absence of the rete in mouse deer provides new insights into the evolution of the arterial system in artiodactyls.

Hair length in mammals is generally regulated by the hair cycle, and its disruption leads to abnormal hair morphogenesis in several species. FGF5, one of the hair cycle regulators, has a role in inducing catagen, and that mutation causes abnormal hair length in both sexes in humans, mice, dogs, and cats. Male-dominant long-haired coat (MALC) is an inbred strain of Syrian hamster exhibiting spontaneous long hair in males. After castration, MALC exhibited significantly shorter hair than the control individuals, but testosterone administration to castrated MALC showed reversion to the original phenotype. Moreover, flutamide administration led to MALC phenotype repression. Histological analysis revealed that hair follicle regression was shown in the wild-type 4 weeks after depilation, but that of MALC remained in the anagen phase. We detected a c.546delG of Fgf5 in MALC (Fgf5malc) that might lead to truncation resulting from a frame shift in FGF5 (p.Arg184GlyfsX6). Additionally, homozygous Fgf5malc was only detected in long-haired (Slc:Syrian×MALC)F2 and (J-2-Nn×MALC)F2 progenies, and all homozygous wild and heterozygous Fgf5malc individuals showed normal hair length. Thus, Fgf5malc leads to male-dominant long hair via a prolonged anagen phase which is affected by testosterone in hamsters. To our knowledge, this report is the first to present the sexual dimorphism of hair length caused by the Fgf5 mutation.

The interaction between vimentin and plectin was examined by immunoprecipitation. The spatial correlation between vimentin, plectin and F-actin in BCa cells was analyzed by confocal microscopy. To evaluate the functions of invadopodia, cells were assayed for extracellular matrix (ECM) degradation, Matrigel invasion, transendothelial invasion and lung metastasis. RESULTS: In invasive BCa cells, the expression levels of vimentin and plectin were elevated and the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of ECM degradation, Matrigel invasion, transendothelial invasion and metastasis. CONCLUSIONS: Our results strongly suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular step for BCa cell invasion and extravasation for metastasis.

Summary The considerable phylogenetical differences between mouse deer and other ruminants have been established by means of DNA sequence analysis and anatomical observations. To clarify the physiological role of the uteroplacenta of the mouse deer, immunohistochemical observation was attempted by using GRP, which has been suggested as a novel regulatory peptide in the female reproductive tract, as an indicator to compare with other ruminants. Strong positive reactions for the GRP were detected in the uterine glands of the pregnant animals, but not in the non-pregnant ones. Although the placenta of the mouse deer is categorized as a diffuse placenta that is different from other ruminants’ polycotyledonary placenta, in terms of GRP immunoreactivity, the mouse deer placenta can be classified as a synepithecholial placenta like the other ruminants’. The secretion of GRP from the uterine glands is of some importance to the fetus in the mouse deer.

Summary The transition of fetal haematopoietic sites in mice was examined histologically from 12.5-day embryos (E12.5) until 10 days of age (D10), and the expression of the adhesive molecules VCAM-1 and fibronectin was examined immunohistologically. Erythropoiesis occurred in the liver (E12.5–18.5), spleen (E18.5–D4) and bone marrow (D6–10), in that sequence. Even at D10, some erythropoiesis occurred in the liver, and more so in the spleen, although the active haematopoietic site was the bone marrow. Similarly, granulopoiesis of neutrophils occurred in the liver, spleen and bone marrow in turn. Granulopoiesis still occurred in the spleen at D10, but no neutrophils were found in the liver after D4. VCAM-1 appeared in the liver, spleen and bone marrow in parallel with active erythropoiesis and granulopoiesis. The co-expression of VCAM-1 and fibronectin was recognized in the endothelial cells of the sinus at the onset of haematopoiesis. This study showed that haematopoiesis in the liver, spleen and bone marrow overlapped peri-natally, although it shifted sequentially. VCAM-1 appears to be closely associated with erythropoiesis and granulopoiesis, and the co-expression of VCAM-1 and fibronectin plays a role in inducing haematopoietic stem cells to move to the tissues.

To reveal the mechanisms regulating the selective atresia of follicles in porcine ovaries, we examined the changes in the mRNA and protein levels of cell-death ligand, Fas/APO-1/CD95 ligand (FasL), and its receptor, Fas/APO-1/CD95 (Fas), and the localization of the proteins in granulosa cells during follicular atresia using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, respectively. Trace levels of FasL mRNA and protein were detected in the granulosa cells of healthy follicles; however, weak levels were detected in those of early atretic follicles, and the levels increased during atresia. Trace/weak levels of Fas mRNA and protein were detected in the granulosa cells of healthy follicles. Fas protein was located in the cytoplasmic area, not in cell membrane area, indicating that it has no activity in regard to inducing apoptosis. When apoptosis commences in granulosa cells, Fas moves from the cytoplasmic to cell membrane area. FasL and Fas mRNAs and proteins in granulosa cells were upregulated during follicular atresia. The FasL and Fas system may play a crucial role in the regulation of apoptosis in granulosa cells during selective follicular atresia in porcine ovaries.

Summary Fibre connections of the chick nucleus geniculatus lateralis ventralis (GLv) were investigated using the axonal tracing method with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the GLv, many labelled neurons were observed in layer i of the stratum griseum et fibrosum superficiale (SGFS) in the ipsilateral tectum opticum (TO) and in the nucleus lentiformis mesencephali (LM). In the TO-GLv projection, cells of origin were located in the deeper part of layer i of the TO and were topographically distributed along the direction from the rostrodorsal part to the caudoventral part of the TO relating to a rostrocaudal axis of the GLv. In the LM-GLv connection, the dorsal and ventral parts of the LM connected reciprocally with the rostral and caudal halves of the GLv, respectively. In contrast, in the GLv efferent connection, labelled axon terminals spread widely in the ipsilateral area pretectalis without any clear topographical arrangement.

Mouse and vole embryos were allogeneically and xenogeneically transferred into pseudopregnant CD-1 and immunodeficient (scid) female mice, and we investigated the distribution of immunocompetent cells, uterine natural killer (uNK) cells, mast cells and macrophages, in the implantation sites on days 6, 7 and 8 of gestation. The survival rate of the vole embryos decreased gradually with increased gestation, but the rate was higher in the scid uteri than in the CD-1 mice. The number of uNK cells increased markedly at the mesometrial triangle and the outer decidual area in the CD-1 uteri containing vole embryos; by contrast, scid uteri having vole embryos showed almost the same number as those having mouse embryos. Mast cells were present in large numbers at the myometrium, but rarely in the decidua in all types of pregnant uteri. Cells at the myometrium were more numerous in xenogeneic than in allogeneic transfer. Many mast cells appeared in the inner decidua where xenogeneically transferred vole embryos were dead and aborted. Macrophages were present in the outer decidua and myometria in all types of pregnant uteri, and their distribution pattern did not change even in aborted uterine sites. These results suggest: (1) the response of macrophages to dead embryos is completely inhibited, (2) uNK cells and mast cells increase near dead and aborted embryos, and (3) the increment in uNK cells responding to xenogeneic embryos is suppressed in scid mice, and the suppression may contribute partly to survival of the embryos.

Mouse deer is known as the smallest and one of the most primitive ruminants. The lesser and the greater mouse deer originated in three different parts of Malaysia were examined for cytogenetic study. The chromosome number of the mouse deer was 2n=32, consisted of 15 metacentric autosome pairs and X and Y sex chromosome pair. G-, R-band revealed polymorphisms of sex chromosomes among the mouse deer from different origins. X chro- mosome varied in length of short arm between the lesser and the greater mouse deer. Y chromosome showed poly- morphisms not only between the lesser and the greater mouse deer, but also among the lesser mouse deer from dif- ferent origins. The 18S-28S rRNA genes (18S-28S rDNA) were localized only in the X and Y chromosomes in both lesser and greater mouse deer by the Fluorescence in situ hybridization (FISH) method. The hybridization signals of 18S-28S rDNA showed polymorphisms not only in size, but also in their distribution patterns on the sex chromo- somes. In addition to the general banding methods, the FISH analysis determined that the greater mouse deer from Tioman island would be remarkably different from the lesser mouse deer. The cytogenetic observations investigated in this study could be informative to characterize the lesser and the greater mouse deer. And it is also suggested that the chromosomal change would reflect that the relationship between the geographic distribution and speciation among the mouse deer.

We examined the phylogeographic structure of the Lesser Mouse deer (Tragulus javanicus) and the Greater Mouse deer (Tragulus napu) from sequence data of mitochondrial cytochrome b gene. Three major clusters were observed on phylogenetic trees, as follows: 1) T. javanicus from the Asian mainland (Malayan Peninsula and Laos), 2) T. javanicus from Borneo Island (Sabah, Malaysia), 3) T. napu from Borneo and Pulau Tioman. Tragulus napu forms the sister lineage to all populations of T. javanicus, thereby supporting the current separation of these taxa at species level and traditional taxonomical identification of the two species by throat color. Within T. javanicus, the primary genetic separation is between the Borneo Island population and those from the Southeast Asian mainland. We suggest that subspecies should be recognised within this widespread taxon. A secondary separation within T. javanicus is observed between populations to the north and south of the Isthmus of Kra.

We examined the geographical variation of the skull size and shape of the lesser mouse deer (Tragulus javanicus) from Laos, Thailand, Peninsular Malaysia, Sumatra, Java, Borneo, Langkawi and some Islands of Tenasserim in Myanmar. Although the influence of the climatic condition on skull size was not confirmed in the mainland populations, the skull became rostro-caudally longer in the populations of Tenasserim and Sumatra because of island isolation effect. The skull size was classified into the following three clusters of localities from the matrix of Q-mode correlation coefficients: 1) Langkawi and Tenasserim, 2) Laos and Thailand, 3) Sumatra and Borneo. The skulls in the population of Java belong to the cluster of Langkawi and Tenasserim in male, however were morphologically similar to those in the cluster of Borneo and Sumatra. The canonical discriminant analysis pointed out that the Laos and Tenasserim populations were separated from the other ones and that the populations of Sumatra, Java and Borneo were intermingled each other.

The skull adaptation was functional-morphologically examined in 14 species of the tree shrews. From the data of the proportion indices, the similarities were confirmed between T. minor and T. gracilis, T. tana and T. dorsalis, and T. longipes and T. glis. We demonstrated that the splanchnocranium was elongated in terrestrial T. tana and T. dorsalis and shortened in arboreal T. minor and T. gracilis from the proportion data. In both dendrogram from the matrix of the Q-mode correlation coefficients and scattergram from the canonical discriminant analysis, the morphological similarities in the skull shape suggested the terrestrial-insectivorous adaptation of T. tana and T. dorsalis, and the arboreal adaptation of T. minor and T. gracilis. Since the osteometrical skull similarities were indicated among the three species of Tupaia by cluster and canonical discriminant analyses, the arbo-terrestrial behavior and its functional-morphological adaptation may be commonly established in T. montana, T. longipes and T. glis.

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene-induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene-induced transgenic mice. The uteri containing xeno-allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid-Schiff-positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.

Reproductive features of newly bred Russian voles (Microtus rossiaemeridionalis) as a laboratory animal were studied. This species is a copulatory ovulator, and most couples copulated 6 to 16 h after pairing. The gestation period varied from 18 to 22 days (mean +/- SD: 20.6 +/- 0.9, n = 72), and the average litter size was 4.6 +/- 1.9 (n = 125). Compared with the litter size at the first parturition (3.6 +/- 1.6, n = 72), the litter size in the subsequent parturitions increased to 5.9 +/- 1.4 (n = 53). The animals exhibited postpartum estrus, and repeated pregnancy accompanied with suckling pups and parturition continuously in the laboratory condition unlike other vole species. In view of their complex stomach and good proliferation, the Russian voles were evaluated as a good laboratory animal, especially as a model animal for ruminant studies.

Skull size and shape were examined among 14 species of the tree shrews (Tupaia montana, T. picta, T. splendidula, T. mulleri, T. longipes, T. glis, T. javanica, T. minor, T. gracilis, T. dorsalis, T. tana, Dendrogale melanura, D. murina, and Ptilocercus lowii). The bones of face were rostro-caudally longer in T. tana and T. dorsalis, contrasting with T. minor and T. gracilis, D. melanura, D. murina and P. lowii which have smaller facial length ratios. The arbo-terrestrial species (T. longipes and T. glis) were similar to terrestrial species in length ratios of bones of face unlike the other arbo-terrestrial species (T. montana, T. picta, T. splendidula, and T. mulleri). We propose that T. longipes and T. glis have adapted to foraging for termites and ants as have T. tana and T. dorsalis. Additionally small body size in T. javanica may be the result of being isolated in Java. We separated the species into 5 groups from the measurment values of skulls: 1) Terrestrial species; T. tana and T. dorsalis, 2) Arboreal species; T. minor and T. gracilis, 3) Arbo-terrestrial species group 1: T. montana, T. splendidula, T. picta and T. mulleri, and T. javanica, 4) Arbo-terrestrial species group 2: T. glis and T. longipes, 5) Arboreal species of Dendrogale and Ptilocercus. Principal component analysis separated species into 8 clusters as follows: 1) T. tana, 2) T. dorsalis, 3) T. montana, T. splendidula, T. picta and T. mulleri, 4) T. glis and T. longipes, 5) T. javanica, 6) T. minor and T. gracilis, 7) D. melanura and D. murina, and 8) P. lowii. We suggest that these clusters correspond to behavioral strategies and peculiarities observed in foraging, feeding and locomotion in each species.

We describe an unusual case of metastasis of the skull base from prostate cancer. A 64-year-old man was referred for progressive cranial nerve palsies. Brain magnetic resonance imaging showed a metastatic tumor in the right petrous bone. He was presenting Garcin syndrome indicating involvement of the right III, IV, V, VI, VII, VIII, IX, X, and XII cranial nerves. The serum PSA level was 5118 ng/mL (standard threshold 4 ng/mL). A transrectal needle biopsy was performed, and poorly differentiated adenocarcinoma was detected in the prostate. Therefore, he was finally diagnosed with skull base metastasis of the prostate cancer. Androgen deprivation therapy and external beam radiation therapy to the skull base resulted in complete resolution of the cranial nerve palsies for more than 30 months. This is the first reported case of metastatic prostate cancer presenting as Garcin syndrome.

INTRODUCTION AND OBJECTIVES: Lysine-specific demethylase 1 (LSD1), the first discovered histone demethylase, is a novel target for prostate cancer therapy. LSD1 expression positively correlates with prostate tumor malignancy. NCL1, a selective cell-active inhibitor of LSD1, was discovered at our university. Here, we verified the mechanism of prostate cancer growth and examined the anti-cancer effect of NCL1 using an animal model. METHODS: We used the prostate cancer cell lines LNCaP, PCai1, and PC3. LSD1 expression was examined by western blotting in LNCaP, PCai1, and PC3 cells. Cell viability was assessed by a WST-1 assay in LNCaP, PCai1, and PC3 cells treated for 48 h with vehicle or 1e100 mM NCL1. LNCaP cells were incubated with or without 30 mM NCL1 for 210 min. Chromatin was collected, and ChIP and real-time polymerase chain reaction were used to confirm the methylation status of the promoter regions of certain genes. Nude mice were subcutaneously injected with 3 106 PCai1 cells. Four days later, mice were injected intraperitoneally with NCL1 at doses of 0.5 mg/kg or 1.0 mg/kg. Mice were treated twice a week for 8 weeks. RESULTS: LSD1 was expressed in all cell lines. The number of viable cells were reduced after NCL1 treatment in all cell lines in a dose-dependent manner. On ChIP analysis, NCL1 treatment induced H3K9me2 accumulation at the androgen response element 1/2 (ARE1/2) and at ELK4 and KLK2 promoter regions. Tumor volume was significantly lower in mice treated with 1.0 mg/kg NCL1 than in controls. Mice that received 1.0 mg/kg NCL1 showed vacuolar degeneration, low Ki67 positivity, and high TUNEL intensity. There were no adverse effects in terms of body weight, relative organ weight, histological findings, and blood biochemical examination results. CONCLUSIONS: LSD1 is involved in prostate cancer cell proliferation via H3K9me2 demethylation at ARE1/2 and ELK4 and KLK2 promoter regions. NCL1 is safe and effective in vivo and induces an anti-cancer effect through apoptosis induction. NCL1 is promising for the development of therapeutics for prostate cancer.

Aniline hydrochloride (AH), a methemoglobin-formation stimulating substance, at a dosage level that does not induce apparent fetal death was injected subcutaneously into pregnant rats once a day on days 6–8, 9–11, 12–14 or 15–17 of gestation in order to assess its ability to stage-specifically produce cardiovascular malformations. In addition, AH at dosage levels of 195, 260, 325 and 395 rag/kg was injected into pregnant rats subcutaneously once a day on days 12–14 of gestation, and the dose-dependent induction of ventricular septal defect (VSD) in relation to maternal methemoglobinemia was studied. In the stage-specificity study, paleness, decreased body weight gain and elevated methemoglobin concentration were noted in the dams. Upon fetal examination, reduced body weight was noted in all AH-treated groups. AH induced cardiovascular malformations, mainly VSD, which was most frequently observed in the day 12–14 group and also observed in the day 15–17 group. Abnormal branching of subclavian, pulmonary and vertebral arteries were most frequently observed in the day 9–11 group. In the dose-dependency study, AH induced maternal methemoglobinemia, decreased fetal body weight and increased the incidence of VSD dose dependency. Additionally, administration of methylene blue, a methemoglobinemia-preventing substance, to the AH-treated dams ameliorated maternal methemoglobinemia and reduced the incidence of fetal VSD. From these results, it is considered that AH stage-specifically induces cardiovascular defects, mainly VSD, in rats and that VSD is induced not by a direct teratogenic effect of AH but by maternal hypoxia due to methemoglobinemia.

Aniline hydrochloride (AH), a methemoglobin formation-stimulating substance, at a dosage level of 520 mg/kg which does not induce apparent fetal death, was injected subcutaneously into pregnant rats once on day 14, 15 or 16 of gestation in order to assess the stage specificity of cleft palate induction. Also, doses of 260, 390, 520 and 650 mg/kg were administered to pregnant rats on day 15 of gestation, and the dose-response relationships with respect to fetal cleft palate and maternal methemo-globinemia induction were studied. In the stage-specificity study, paleness, decreased body weight gain and elevated methemoglobin concentration were noted in the dams treated with AH. Upon fetal examinations, although reduced body weight was noted in all AH-treated groups, cleft palate was observed only in fetuses from those dams treated on day 15 of gestation. In the dose-dependency study, AH induced maternal methemoglobinemia, decreased fetal body weight and increased the incidence of cleft palate dose dependently when administered at dosage levels of 260, 390, 520 and 650 mg/kg on day 15 of gestation. Additionally, administration of methylene blue, a methemoglobinemia-preventing substance, to the AH-treated dams ameliorated maternal methemoglobinemia and reduced the incidence of fetal cleft palate. In summation, it is considered that AH stage-specifically induces cleft palate in rats and that cleft palate is caused not by a direct teratogenic effect of AH but by maternal hypoxia due to methemoglobinemia.

Distribution pattern of the vagal preganglionic neurons in the dorsal motor nucleus of the vagus nerve (DMN) and nucleus ambiguus (NA) projecting into the stomach of the Japanese field voles (Microtus montebelli) was examined using horseradish peroxidase (HRP). The HRP was injected into ten different stomach regions covering the forestomach, glandular region and pyloric stomach. The labeled neurons in the DMN were in every injected case, while labeled neurons in the NA were in the HRP injected into the forestomach and pyloric stomach. After a separate injection of HRP into the ventral and dorsal stomachs, the labeled neurons were predominant in the left and in the right DMNs, respectively. Medio-lateral and rostro-caudal distribution of HRP-labeled neurons showed that many of them were in the medial part and near the level of the obex in the DMN. Especially innervating neurons of glandular region were remarkably restricted in the medial part and at the level of the obex in the DMN. The present investigation showed that majority of neurons supplying each stomach region were in the DMN with site specific and topographic arrangement, and a considerable number of labeled neurons were distributed symmetrically in the NA.

The distribution in the chicken of motoneurons innervating the hyolingual muscles, i.e. the M. branchiomandibularis (BM), M. ceratoglossus (CG), M. interceratobranchialis (CB), M. serpihyoideus (PH), M. stylohyoideus (YH) and one of the mandibular muscles, M. depressor mandibulae (DM), was examined by retrograde transport of horseradish peroxidase conjugated with wheatgerm agglutinin. Labelled motoneurons are found in the three subnuclei of the facial (VII) nucleus, as well as hypoglossal (XII) nucleus. The distribution of motoneurons projecting to the DM are observed in the three subnuclei, those of the BM, PH and YH in the intermediate and ventral subnuclei and those of the CB and CG in the intermediate subnucleus. Motoneurons projecting to the PH, YH, CB and CG are also distributed in the XII nucleus. The ratio of labelled motoneurons of the VII to XII nuclei decreases the PH, YH, CB and CG in that order, and the ratio of labelled ones of the ventral to intermediate subnuclei decreases the BM, PH and YH in that order. The topographical and functional aspects of the subdivision of the motor nucleus are discussed.

We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.

The semidominant gene Dh (Dominant hemimelia) induces skeletal and visceral abnormalities of various degrees and failure of the spleen in mice. The homozygous individual (Dh/Dh) seems to be lethal. The present experiment was designed to investigate the ability Dh cells to form a spleen and the genesis of the hind limb malformations by Dh/Dh and Dh/+ cells in chimeric mice. The Dh/Dh and Dh/+ embryos were produced in the F2 progeny of a cross between inbred strains of Dh/+ and DDD mice. They were aggregated with C3H/He or C57BL/6 embryos to make chimeras. Identification of Dh/Dh or Dh/+ embryos was carried out by Pep-3, and chimerism was analyzed by Gpi-1. Of 25 chimeras carrying the Dh gene, four mice formed a small spleen, two mice had a vestigial spleen, and the others no spleen. The tissues of the incompletely developed spleens were normal histologically and Dh cells were involved in the tissues of the spleen. In the chimeric mice, hindlimb malformation by the Dh gene was reduced in severity and the lethality of the homozygote (Dh/Dh) was rescued.

Retinal projections in the Japanese field vole (Microtus montebelli) were determined by anterograde transport of horseradish peroxidase (HRP). Injection of HRP into the unilateral vitreous body demonstrated that the terminal labeling of the optic projections was seen bilaterally in the suprachiasmatic nucleus (SCH), the ventral (GLv) and dorsal (GLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the medial pretectal nucleus (NTOM) of the pretectum (PT) and the superficial layer of the superior colliculus (CS), with contralateral predominance, and only contralaterally labeled terminals were found in the lateroposterior thalamic nucleus (LP), the lateral pretectal nucleus (NTOL) of the PT, the dorsal (DTN) and medial (MTN) terminal nuclei of the accessory optic system (AOS). The distribution area of the retinofugal terminals was divided into a three laminar arrangement in the GLd, i.e., layers 1 and 3 and layer 2, received the retinal input from contralateral and ipsilateral eye, respectively, as in arboreal squirrels. The contralateral CS received retinal fibers in the superficial layer, while ipsilateral optic fibers projected sparsely to the stratum opticum of the colliculi. Retinal connections to the DTN and MTN of the AOS were clearly discerned but no lateral terminal nucleus with retinal afferents was found. In addition, the AOS had no inferior fasciculus. These findings indicate that the vole has a contradictory features of a well- and a less-developed sense of vision. Namely, the image forming visual system such as the retino-GLd was as well-developed as in a squirrel, on the other hand, the non-image forming visual system such as the retino-AOS was less-developed as in an insectivore's brain.

Tetraploid mouse embryos usually cease to develop early after implantation, though they can develop to blastocysts. To characterize the failure of development in detail, tetraploid mouse embryos at the preimplantation period were examined as to both their morphology and number of cells. The tetraploid embryos were produced by 12 hr treatment with cytochalasin B (CB) at the 2-cell stage of backcross of (C57BL/6 x C3H/He) F1 x C3H/He. The tetraploid embryos in the preimplantation period exhibited compaction at 72 hr after hCG injection and blastocyst formation at 96 hr, as well as diploid embryos, but the number of cells composing the embryos was significantly smaller than that in the diploid embryos. At the term 60-96 hr after hCG injection, mean cell cycles were 14.03 hr in the tetraploid embryos, but 12.02 hr in the diploid. When tetraploid embryos were transferred into the oviducts of pseudopregnant recipients immediately after CB treatment, the number of cells in tetraploid blastocysts was increased compared with the embryos cultured in vitro, though the number did not reach that of diploid embryos. These results suggested that compaction and blastocyst formation in preimplantation development of tetraploid embryos depended on the time after hCG injection, irrespective of the number of cells or the length of the cell cycle. The lengthening of the cell cycle in tetraploid embryos may be one of the causes of failure in postimplantation development.

SMXA recombinant inbred (RI) strains of mice were produced by systematic inbreeding from the F2 generation of a cross between two progenitor inbred strains, A/J and SM/J, which differ considerably with respect to many characteristics, and consists of 28 inbred strains. In this study, we investigated the applicability of DNA fingerprinting with M13 phage DNA to the identification of these closely related strains. DNA fingerprints of the SMXA RI strains and their progenitors, SM/J and A/J, showed strain-specific patterns, with the same banding patterns within each strain. Linkage analysis by using strain distribution patterns of minisatellite loci with 108 genetic markers containing microsatellites, biochemical and immunological marker genes allowed 23 minisatellite loci to be assigned to 11 chromosomes. The results suggested that DNA fingerprinting with M13 phage DNA is applicable not only for strain identification but also for genetic monitoring of RI strains on almost all chromosomes.

Leydig cells of lesser mouse deer (Tragulus javanicus) testes were observed using light and transmission electron microscopies. Sexually mature lesser mouse deer were obtained in East Malaysia. The testes were perfused with 5% glutaraldehyde, postfixed with 1% OsO4, dehydrated in ethanol and embedded in Araldite. The semithin sections were cut, stained with toluidine blue and observed under light microscopy. The ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a JEM-1200 transmission electron microscope. As a result, two types of filament bundles were frequently recognized in Leydig cells, but not in other testicular cells. These bundles were clearly seen at even a light microscopic level. One type was bundles of actin filaments (approximately 5 nm in diameter). These structures were found not only in the cytoplasm but also in the nucleus. The other type was bundles of intermediate filaments (approximately 10 nm in diameter). These structures were found only in the cytoplasm. The existence of filament bundles has never been reported in the testicular cells of another mammalian species. Thus, while bundles of actin and intermediate filaments are specifically present in the Leydig cells of the lesser mouse deer, their functions are still unclear.

Body fluids include blood, lymph, tissue fluid, milk and saliva. They are produced in the body, then circulated within it or secreted outside it. Several kinds of body fluids are collected and analysed to check the health status of animals. In particular the blood is examined periodically to monitor microbial contamination and genetic contamination from other strains. Moreover, body fluids are used in many kinds of research for various purposes. The availability of body fluids from mice is limited because of their small quantity. In this chapter procedures to collect body fluids available from mice, such as blood, urine, milk, bile, semen, saliva, lacrimal fluid and peritoneal fluid, are described in detail.

Thirteen days after capture, the blood serum of eight anesthetized female leaf-monkeys,Presbytis cristatus (3.86 kg of mean body weight), were analyzed for hematocrit (35.6±6.7 %), total protein (6.7±0.8 g/dl), albumin (3.61±0.77 g/dl), α-1 globulin (0.13±0.04 g/dl), α-2 globulin (0.73±0.18 g/dl), β globulin (0.87±0.27 g/dl), γ globulin (1.36±0.55 g/dl), A/G ratio (1.23±0.38), Na (161±6.14 mEq/l), K (5.61±0.74 mEq/l), LDH (575±257 IU/l), GOT (93±67 IU/l), GPT (34±33 IU/l), CPK (250±200 IU/l), ALP (613±633 IU/l), LAP (115±68 IU/l), γ-GTP (28±30 IU/l), TG (47±24 mg/dl), T-Cho (141±31 mg/dl), BUN (29.0±5.7 mg/dl), T-bil (0.21±0.07 mg/dl), and IP (3.4±2.0 mg/dl).

Identification of substrains or sublines of inbred mice and assessment of genetic relationships among them were performed on the basis of DNA fingerprinting using M13 phage DNA as a probe. We used eight C57BL/6 sublines (J parallel Jcl, J parallel Jms, J parallel Slc, J parallel Nrs, J parallel Yok, Jah, N parallel Crj, N parallel Jcl) and eleven C3H/He sublines, (J, J parallel Jcl, J parallel Yok, J parallel Nrs, J parallel Jms, N, N parallel Jcl, N parallel Crj, Slc, Jah, Nrs). Two kinds of restriction endonucleases (HinfI and PstI) were used. It was found that: 1) DNA fingerprint within each subline showed identical patterns. 2) Most sublines of C57BL/6 and C3H/He could be identified using DNA fingerprinting with HinfI except between N parallel Crj and Slc, and among J, J parallel Nrs and J parallel Yok in C3H/He. DNA fingerprints with PstI endonucleases showed low polymorphic banding patterns. 3) A dendrogram constructed from DNA fingerprint patterns reflected generally the genealogy of the sublines used. 4) DNA fingerprinting, therefore, seemed to be suitable for the genetic monitoring and assessment of genetic relationships among sublines of inbred mice having close relationships.

Protrusion of hyperplastic upper molar teeth (macrodonts) into the cranial cavity was observed in the Japanese field vole. Protrusions of the upper molars occurred on both sides, but were large on the left than on the right. The teeth invaded the cranial cavity and brain parenchyma. Large concavities on the ventral side of the brain were interposed between the lateral olfactory tract and the optic tract. Invation by one macrodont reached the basal nuclei, hypothalamic and thalamic nuclei. The brain was deformed bilaterally, and nearly the entire olfactory bulb, globus pallidus, internal capsule, and lateral preoptic nucleus, and a portion of the hypothalamus, thalamus and anterior commissure were absent on the left side.

Features of a long-haired mutant and inheritance of mutation were investigated in the golden hamster (Mesocricetus auratus). The hair of adult long-haired hamsters was longest at the rump and flanks (approximately 70 mm) and was also unusually long at the neck (45 to 50mm). The hair length of males was more striking than that of females. Mating experiments indicated that an autosomal recessive gene is responsible for the inheritance of long hair. An albino long-haired hamster strain was established by continuous full-sib mating from F2 hybrids, descendants of (acromelanic albino x agouti long-haired) F1 progeny, and genetic homogeneity was confirmed by DNA fingerprinting. The long-haired strain was characterised by the fact that males grew larger than females, in contrast to the other strains of hamsters.

Adrenocortical zonation in the laboratory Japanese field vole (Microtus montebelli) was investigated histologically with special attention to the sex and species specific differences. The whole cortex and zona reticularis showed clear sex differences. They were thicker in females than in males. In this respect the vole was similar to the mouse and rat, but different from the Syrian hamster and mastomys. Male cortices were characterized by thick z. fasciculata and meagre z. reticularis, while both zones were thick in females. The z. fasciculata in both sexes and z. reticularis in females seemed to be divided into two sublayers by the cellular arrangement. No distinctive additional zones were identified. The histological observations indicated that the Japanese field vole might have interesting endocrinological characteristics and serve for studying the mechanism of the zone formation in the mammalian adrenal cortex.

OBJECTIVES To establish a novel and simple method of preventing post-retropubic prostatectomy (RRP) inguinal hernia. Inguinal hernias occur in 8%-22% of men within 1-2 years of RRP. Although manipulation during RRP might weaken the normal fascia structure at the internal inguinal ring with the vas deferens, the exact mechanism of post-RRP inguinal hernia remains unknown. METHODS Several surgeons performed RRP at our hospital on 271 patients between April 2004 and September 2009. Among these patients, post-RRP measures to prevent inguinal hernia were applied to 101 patients (group A) and not applied to 170 patients (group B). We released the bilateral spermatic cord from the peritoneum before suturing the wound, which should prevent the intestinal tract coated with the peritoneum from pushing through the internal inguinal tract. We compared the incidence of postoperative inguinal hernia between the 2 groups. RESULTS The patients were followed up for an average of 11.6 (range: 2-22 months) and 23.9 (range: 23-24 months) months in groups A and B, respectively. Inguinal hernia developed in no patients in group A and in 20 (11.8%) in group B. The hernia-free rate was significantly lower in group B than group A. All postoperative inguinal hernias were indirect. The median interval between surgery and hernia diagnosis was 10.6 months (range, 2-24), and 16 patients (80%) were diagnosed within 12 months. CONCLUSIONS We developed a simple method of preventing inguinal hernia after RRP. Our technique is simple enough to complete within a few minutes, and the outcome is excellent.

The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d-galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N-acetyl-d-galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N-acetyl-D-glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), D-mannose and d-glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), L-fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.