Comparison of the gut microbiota of captive common bottlenose dolphins Tursiops truncatus in three aquaria.

AIMS: This study was conducted to assess the presence and extent of differences in the gut microbiota of common bottlenose dolphins depending on rearing facilities. METHODS AND RESULTS: Faecal samples were collected from 16 common bottlenose dolphins at three aquaria in Japan. After extracting DNA from the faeces, the V3-V4 region of bacterial 16S rRNA was amplified and sequenced using Illumina MiSeq platform. The constituent phyla of the gut microbiota were similar among aquaria; however, the most dominant phylum differed depending on the facility, and the compositions of microbiota were remarkably varied at the family level among aquaria. The alpha diversity indices tended to differ among aquaria. Some bacterial families observed in terrestrial mammalian carnivores or carnivorous fish were detected, as well as several bacterial species suspected of being pathogenic in dolphins. CONCLUSION: Our findings indicate that captive environmental conditions including prey and housing types may contribute to differences in the gut microbiota of the dolphins. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study revealing the differences in gut microbiota of captive dolphins among facilities. Our findings will provide valuable information for improving the health management of dolphins.

Human cord blood cells transplanted into chronically damaged liver exhibit similar characteristics to functional hepatocytes.

Human umbilical cord blood (CB) cells have many advantages as a source for stem cell transplantation because of immaturity and availability. It has been reported that CB cells transplanted into an injured liver displayed hepatocyte-like phenotypes. However, there have been few studies to characterize CB-derived hepatocyte-like cells (HLCs). In this study, CB cells were transplanted into mice with 2 types of liver damage: transient and chronic damage. We analyzed the expression of hepatic differentiation markers in CB-derived HLCs. In the liver of NOD/SCID mice with transient damage, CB-derived HLCs were detected infrequently at 3 weeks after transplantation. In contrast, in the liver of SCID mice damaged chronically by a urokinase-type plasminogen activator transgene under the control of albumin promotor/enhancer (ALB-uPA/SCID mice), more human HLCs colonized the host liver compared with hosts with transiently damaged livers. The CB-derived HLCs in both the transiently and the chronically damaged liver expressed a few markers of human hepatocytes, whereas the transcripts related to mature hepatic functions, including cytochrome P450s, were detected only in the ALB-uPA/SCID mice. These data indicated that CB cells were able to display a similar phenotype to functional hepatocytes in the recipient liver with chronic damage. CB cells may represent a transplantable source for chronic decompensated liver disease.

Impaired circadian rhythm of gastric myoelectrical activity in patients with multiple system atrophy.

In order to evaluate gastric motility and its circadian rhythm in patients with multiple system atrophy (MSA) and healthy control subjects, we measured gastric myoelectrical activity (GMA) for 24 hours using a cutaneous electrogastrogram (EGG) recorder in 14 MSA patients and 9 age-matched controls. We analyzed six 10-minute segments of EGG before and after each meal and two 20-minute EGG segments during sleep; three parameters were used for the analysis: dominant frequency (DF), instability coefficient of dominant frequency (ICDF), and dominant power (DP). DF increased during daytime and decreased during sleep in the control, while this circadian variation was blunted in the patients with MSA. The average DF of the eight segments in the MSA patients did not differ from that of the control. Both MSA patients and control subjects did not show the circadian variation of ICDF and DP. The average ICDF of the eight segments in the patients with MSA was significantly decreased when compared with that of the control (p < 0.01). No differences were observed in DP between the two groups. This study indicates that the healthy subjects appear to have a circadian rhythm of DF, and the patients with MSA appear to have impaired circadian rhythm of DF and decreased ICDF possibly due to the degeneration of the central autonomic neurons.

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies.

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.

Isolation of hepatocyte-like cells from mouse embryoid body cells.

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.

Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells.

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cell activation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoing in vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle alpha-actin, PDGF receptor-beta, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.

Kdap, a novel gene associated with the stratification of the epithelium.

The skin develops and differentiates during embryogenesis, which is concertedly regulated by a variety of genes. The present study isolated from the rat embryonic skin a novel differentiation-associated gene named Kdap (keratinocyte differentiation-associated protein) by suppression subtractive hybridization between the skin of 14day postcoitus (dpc) embryo (the prehair-germ stage) and that of 17dpc embryo (the hair-germ stage). Its mRNA contained four spliced forms in these tissues. The gene encoded a protein of total 98 amino acids with a calculated molecular mass of 11kDa and an isoelectric point of 6.1 as an unspliced form. The two splicing zones were well conserved among rat, mouse, and human. This protein had a high hydrophobic N-terminal region, a possible signal sequence, and contained two putative N-myristoylation sites and two casein kinase II phosphorylation sites. In situ hybridization experiments detected Kdap transcripts exclusively in the suprabasal cell layers of the embryonic epidermis. Intense expression was also seen in suprabasal cells in regions of infundibulum of the hair follicle. These results indicated that Kdap provides a new insight into the mechanism of differentiation and the maintenance of stratified epithelia.

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin.

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.

Expression of genes of type I and type II collagen in the formation and development of the blastema of regenerating newt limb.

We cloned cDNAs of alpha1(I) and alpha1(II) collagen, and studied their expression profiles in regenerating limbs of newts, Cynops pyrrhogaster. The expression of the alpha1(I) gene was markedly up-regulated at the early bud stage of the blastema. In situ hybridization experiments revealed that the alpha1(I) gene was expressed in not only mesenchymal cells of the blastema, but also the basal cells of the wound epidermis at the wound healing stage when the epidermal basement membrane was absent. This unique expression continued until 21 days (late bud stage), while the basement membrane began to form at 14 days. These results indicate biochemical differences between the wound and normal epidermis, and suggest the direct involvement of the former in the synthesis of blastemal matrices of type I collagen. Actually, immunohistochemistry revealed that type I collagen began to be deposited beneath the wound epidermis at 8 days, and accumulated there and around blastemal mesenchymal cells at 14 to 21 days. Undifferentiated mesenchymal cells associated with the amputated muscle fibers actively expressed the alpha1(I) gene. Mesenchymal cells in the central region of blastemas deposited type I collagen fibers around them. Concomitantly with the appearance of prechondrocytes, the alpha1(II) collagen gene became activated. The present study clearly shows that the expression of the genes of both type I and type II collagen in blastemal cells is temporally and regionally well-regulated in a cooperative manner. Dev Dyn 1999;216:59-71.

Cell-type specific and thyroid hormone-dependent expression of genes of alpha1(I) and alpha2(I) collagen in intestine during amphibian metamorphosis.

Both the epithelium and the mesenchyme of the larval small intestine of anurans undergoes metamorphic conversion into the adult counterparts. The conversion of the mesenchyme has been poorly understood especially at the molecular level, whereas the changes of the epithelium have been extensively studied. The present study investigated the metamorphic changes of the mesenchyme of tadpoles of bullfrog, Rana catesbeiana, focusing on the expression of genes of type I collagen. By using the cDNA clones coding for a 1(I) and a 2(I) collagen as probes, expression of each collagen gene was examined. These genes were drastically up-regulated at the climax period of spontaneous metamorphosis, which was precociously mimicked by treating tadpoles with thyroid hormone. The increased expression of these genes at the climax stage was well correlated with the conversion of the thin larval mesenchyme to more thick and dense adult connective tissues of the intestine. In situ hybridization identified the fibroblasts that were actively expressing the collagen genes and, therefore, were thought to be responsible for the remodeling. These results strongly suggest that the expression of type I collagen genes is regulated during the intestinal remodeling in a cell-type specific and thyroid hormone-dependent manner.

Plasma calcium and calcitonin levels in eels fed a high calcium solution or transferred to seawater.

To examine the physiological role of calcitonin (CT) in calcium homeostasis of teleosts, we compared calcium and CT levels in freshwater eels fed a high calcium-consomme solution (Ca2+: 1.25 M; 1 ml/100 g body wt) into the stomach (Experiment I), and in freshwater eels transferred from freshwater to seawater (Experiment II). In experiment I, plasma calcium and CT levels in the high calcium-treated eels rapidly increased (calcium: 2.63 mM at 0 h to 8. 50 mM at 3 h; CT: below detection level at 0 h to 1118.2 pg/ml at 3 h). Plasma calcium and CT levels in the control eels remained below detection level during the 3 h of the experiment. In experiment II, the plasma CT levels did not increase, although the plasma calcium levels increased from 3.23 mM at 0 h to 4.10 mM at 8 h. Therefore, in eels, we demonstrate a correlation between plasma CT and plasma calcium raised by dietary calcium in the consomme form, but it does not participate in the initial processes of seawater adaptation.

Cloning and characterization of the full length cDNA encoding alpha2 type I collagen of bullfrog Rana catesbeiana.

The present study determined nucleotide sequences of the full-length cDNA of alpha2 chain of bullfrog type I collagen. Hybridization of a bullfrog cDNA library with human alpha1 type I collagen cDNA yielded a clone named 6A-1 which was 3449 bp long and lacked a 5' region of the gene. A 5' region containing the translation initiation site was amplified by the reverse transcription polymerase chain reaction using poly(A)+RNA from tadpole tail tissues as template, and oligonucleotides encoding the translation initiation region of mammalian fibrillar collagens and the Gly-X-Y repeat region of clone 6A-1 as primers. As a result we obtained a 1518 bp long clone Y31. A 355 bp long clone Y31-9 was produced by extending clone Y31 from its ATG codon to a 127 bp upstream region. Combining these three clones, the complete nucleotide sequence of the full-length cDNA was determined which contained 4692 bp as a whole and 4065 bp in the open reading frame. The comparison of its structure with known collagen cDNAs of various vertebrates showed that the cDNA obtained codes for alpha2(I) chain of bullfrog. Its deduced amino acid sequence revealed the complete conservation of seven cysteine residues in the C-propeptide and three lysine residues in the N-telopeptide through the helical domain. Northern blot analysis revealed that the thyroid hormone regulated the expression of alpha2(I) collagen in an organ-dependent manner: intense up-regulation in the back skin and intestine, weak and transient up-regulation in the liver, and initial down-regulation, but later up-regulation in the tail. Prolactin increased its expression in both the back skin and tail. These results suggested that the expression of bullfrog alpha2(I) collagen is cooperatively regulated by these two metamorphosis-regulating hormones.

17 alpha,20 alpha-Dihydroxy-4-pregnen-3-one is the naturally occurring spermiation-inducing hormone in the testis of a frog, Rana nigromaculata.

Spermiation, the process in which vertebrate spermatozoa are detached from investing Sertoli cells into the lumen of the seminiferous tubule, is a prerequisite for the successful fertilization. Using an in vitro Rana nigromaculata spermiation bioassay, we have shown that gonadotropin initiates spermiation by inducing the synthesis of delta 4-steroids by testis fragments. Among all of the delta 4-steroid metabolites produced by R. nigromaculata testis fragments, spermiation-inducing activity was confined to only one metabolite; this metabolite was identified as 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17 alpha,20 alpha-DP). Induction of spermiation by gonadotropin in vitro was accompanied by marked elevations in 17 alpha,20 alpha-DP concentrations in incubation media. These findings provide evidence that 17 alpha,20 alpha-DP is the nautrally occurring spermiation-inducing hormone in R. nigromaculata.

Self-expanding stainless steel stent application in rectosigmoid stricture.

In recent years, several reports on the experimental and clinical applications of the Gianturco stent (self-expanding stainless steel stent) have been published. However, to our knowledge, the use of stents in rectosigmoid strictures has not been reported. We used self-expanding stainless steel stents to dilate rectosigmoid strictures caused by nonresectable recurrent neoplasm. Insertion and dilation (sigmoid colon and rectum) in two patients were successful. Accordingly, these patients were able to maintain bowel activity and avoid palliative loop colostomy. We believe that this procedure is effective for nonresectable rectosigmoid stricture due to recurrent neoplasm.

Biosynthesis of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one in the ovaries of grey mullet (Mugil cephalus) during induced ovulation by carp pituitary homogenates and an LHRH analogue.

Three female mullets received a priming injection of carp pituitary homogenate followed by a resolving injection of an LHRH analogue 24 hr later. Ovarian biopsies were obtained just prior to the first injection (phase I), 24 hr after the first injection (i.e., immediately before the second injection, phase II), and 8 hr after the second injection (phase III). Two fish (Nos. 1 and 3) spawned approximately 12 hr after the second injection. Serum levels of testosterone increased to some extent during phase II in all of the fish. Testosterone levels decreased abruptly during phase III in both fish Nos. 1 and 3. In contrast the concentration of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was undetectable in phases I and II, and increased dramatically during phase III in the same fish. In fish No. 2, which did not spawn, neither the decrease of testosterone nor the increase of 17 alpha,20 beta-diOHprog was observed. Ovarian tissues of all the fish were pooled in each phase and incubated with 14C-labeled progesterone or 17 alpha-hydroxyprogesterone to investigate steroid metabolism. During phases I and II progesterone was converted to 17 alpha-hydroxyprogesterone, androstenedione, and testosterone. During phase III, production of these steroids decreased drastically, and in turn, synthesis of 20 beta-hydroxy-4-pregnen-3-one and 17 alpha,20 beta-diOHprog was induced. Using 17 alpha-hydroxyprogesterone as a substrate, androstenedione and testosterone were produced during phases I and II, whereas they decreased considerably during phase III. This was followed by the production of 17 alpha,20 beta-diOHprog as the major metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)

[Venous aneurysm of V. saphena accessoria--a case report].

A 43-year-old male was admitted for subcutaneous mass of rt. inguinal lesion. Saccular dilatation of vein is a rare phenomenon and usually termed "venous aneurysm". Venous aneurysm has been reported in the following vein: face, neck, extremities, superior vena cava region, portal vein region and by-pass grafting vein. This is the 1st report of a venous aneurysm found in V. saphena accessories in Japan. The true etiology is unknown, but underlying causes which produce damage to the venous wall (trauma, inflammation, congenital weakness, and localized degenerative change) have been mentioned as possible causes. Rarely, thrombosed venous aneurysm may cause pulmonary emboli. Definitive diagnosis is established by venography. Venous aneurysm often increases or reduces in size according to the change of venous pressure by proximal vein compression postural change and Valsalva maneuver. In general, surgical resection of the aneurysm is the treatment of choice.

[Experience of surgical therapy of pyothorax in the pleural fistula of the bronchi and esophagus due to esophageal cancer].

The esophagectomy and total left pneumonectomy were carried out in a 53-year-old male who had developed pyothorax in the pleural fistula of the bronchi and esophagus due to esophageal cancer, and then reconstruction of the esophagus by route of the anterior thoracic wall was performed using stomach tube biphasically. The postoperative course was favorable, and oral intake was possible, and his physical condition recovered as the patient could repeat stopping out from hospital while he died by pneumonia after 7 months. The remote metastasis or metastasis to the mediastinal lymph-node were not noted by autopsy. It is considered that there are cases having indication for active resection among A3-esophageal cancers.

Spectrophotometric kinetic studies and substrate inhibition of hog kidney histaminase activity by an improved 2,4-dinitrophenylhydrazine method.

We improved the experimental procedure for the measurement of hog kidney histaminase activity using histamine as a substrate on the basis of a spectrophotometric estimation of the 2,4-dinitrophenylhydrazone of imidazole acetaldehyde and studied the steady-state kinetics to obtain the basic data for further investigations of the oxidative deamination of histamine. The initial and mean velocities of the enzymatic reaction were calculated and plotted against the amount of enzyme. It was found that the initial velocity increased linearly. The time t alpha necessary to reach the extent of reaction alpha was calculated and plotted against the reciprocal of the enzyme concentration eO. It was found that t alpha was linearly proportional to 1/eO. From Lineweaver-Burk plots, inhibition by high concentration of substrate was evident, and the v-pS curve was bell-shaped, with a pS maximum at 3.2. Km and V were obtained: Km = 7.7 x 10(-5) M, V = 0.0026 mumol/min (0.00075 mumol/min/mg protein). It was concluded that our DNP method was useful for the measurement of hog kidney histaminase activity using histamine as a substrate, basic steady-state kinetic studies and further investigations of substrate inhibition and inhibitory effect.

Structure and steroidogenic enzymes of the seminal vesicles of the urohaze-goby (Glossogobius olivaceus).

The in vitro steroid metabolism in the seminal vesicles of the brackish water goby (urohaze-goby, Glossogobius olivaceus) was studied using males in the breeding season. The moderate activity of delta 5-3 beta-hydroxysteroid dehydrogenase was histochemically detected only in the epithelial cells of the organ, though these cells have the characteristics of secretory cells ultrastructurally. Cell-free homogenates (800 g supernatant fluid) of the whole tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, or testosterone in the presence of NAD+ or NADPH. Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product) and 17 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was metabolized into androstenedione (main product) and 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione, and androstenedione. These results indicate that the steroid metabolic patterns in the seminal vesicles of G. olivaceus are closely resembled to those in the testes.