Laparoscopic cholecystectomy using the subserosal layer dissection technique in dogs: 34 cases (2015-2021).

OBJECTIVES: To describe a standardised subserosal layer dissection technique and evaluate its outcomes in canine laparoscopic cholecystectomy. MATERIALS AND METHODS: Medical records of dogs undergoing laparoscopic cholecystectomy using the standardised subserosal layer dissection technique for the treatment of cholecystolithiasis, cholecystitis, and gall bladder mucocele at a single veterinary hospital from January 2015 to September 2021 were extracted. Operative time, subserosal layer dissection achievement rate, open conversion rate, and complication rate were evaluated. RESULTS: Thirty-four dogs were included. The most common preoperative diagnosis was cholecystolithiasis (n=29). Operative time was 190 minutes (range: 110 to 330 minutes). Subserosal layer dissection of more than 90% of the gall bladder bed was achieved in 27 (79%) dogs. Conversion to open surgery was required in three (8.8%) dogs. There were no cases of intraoperative bleeding, bile duct injury, or reoperation. CLINICAL SIGNIFICANCE: This study showed that laparoscopic cholecystectomy using the standardised subserosal layer dissection technique could be performed successfully in dogs. Future prospective clinical studies are needed to determine safety and effectiveness of this technique compared to standard techniques.

Brain tissue water comes in two pools: evidence from diffusion and R2' measurements with USPIOs in non human primates.

Diffusion-weighted MRI of non-human primates revealed that USPIO Bulk Magnetic Susceptibility (BMS) T2' effects of Ultrasmall Superparamagnetic Particles with Iron Oxide (USPIO) in the brain cannot be explained by a single compartment model, as diffusion and T2' effects appear coupled: Apparent Diffusion Coefficient (ADC) values depend on USPIO concentration and relaxivity effects of USPIO decrease with the b value. On the other hand, USPIO and diffusion effects could be well uncoupled using a model consisting in a fast and a slow diffusion pool with different relaxivities. Diffusion-weighting acts as a filter which emphasizes the contribution of the slow pool when increasing b values (apparent decrease in ADC and R2'). Those results have implications for human studies using BMS contrast agents, as well as BOLD and diffusion fMRI.

Introduction and evaluation of computer-assisted education in an undergraduate dental hygiene course.

OBJECTIVE: This paper introduced newly developed computer-assisted learning materials and reports of a survey of junior college dental hygiene students who have used them. METHODS: We authored new educational material to promote students' basic dental hygiene practice skills using a simulation software generator. A set of five developed materials were tested by 43 female second-year dental hygiene students during the second semester at a college in Chiba, Japan. The evaluation was conducted in the form of a questionnaire including open-ended questions. Students' opinions were analysed using characteristic diagrams, a troubleshooting tool that can be used to visually illustrate the causes and effects of a problem. RESULT: The overall results of the evaluation were positive. The students were given five sets of simulation learning materials (SLMs). Eighty-three percent of the students felt that they could carry out independent study of clinical practice better after the virtual practice. Ninety-three percent of them felt that the exercises should be continued in the future, and eighty-eight percent of them felt that this virtual practice deepened their interest in other classes and training sessions. All of the students found the virtual practice beneficial for their learning. DISCUSSION: The present results suggest that the students became conscious of their lack of knowledge through SLMs. These findings indicate that SLMs for practicing basic clinical procedures is beneficial.

Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II.

The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.

Inhibition of transcription elongation by the VHL tumor suppressor protein.

Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, Elongin (SIII), is identified as a functional target of the VHL protein. Elongin (SIII) is a heterotrimer consisting of a transcriptionally active subunit (A) and two regulatory subunits (B and C) that activate transcription elongation by RNA polymerase II. The VHL protein was shown to bind tightly and specifically to the Elongin B and C subunits and to inhibit Elongin (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.

Induction of apoptosis and cellular senescence in mice lacking transcription elongation factor, Elongin A.

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.

Modification of LSC spectra of 125I by high atomic number elements.

The 125I pulse-height spectra via a liquid scintillation counter (LSC) displayed notable variations. The counting efficiencies of higher and lower energy peaks increased and decreased, respectively, with the enhancement of the amount of high atomic numbered elements within the cocktails. This tendency was ascribed to the increasing probability of the interaction of photons with the scintillation cocktail. Moreover, it was noted that the shape of a 125I spectrum strongly depends on the amount of high atomic numbered elements.

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

Identification of arginase in human placental villi.

l-Arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts l-arginine to urea and ornithine, which is the principal precursor for production of polyamines required for cell proliferation. Human placenta expresses endothelial NOS (eNOS) in syncytiotrophoblasts, but the expression of arginase has not been fully elucidated. Our aim was to investigate the expression and distribution patterns of arginase-I (A-I) and arginase-II (A-II) in human placental villi in the first trimester and at term using immunohistochemistry, RT-PCR and Western blot analysis. The arginase enzyme activity in placental villi was also measured. Immunohistochemistry showed different distribution patterns of the arginase isoforms during gestation: A-I was observed only in cytotrophoblasts, while A-II was observed in both cytotrophoblasts and syncytiotrophoblasts. RT-PCR and Western blot analysis showed expression of A-I and A-II in the first trimester and at term in human placental villi. Expression of A-II and arginase activity was greater in the first trimester than at term. Differentiation of cytotrophoblasts into syncytiotrophoblasts may be associated with l-arginine metabolism through modulation of l-arginine availability for eNOS and A-I. And elevated arginase activity in the early gestational period may be responsible for proliferation of trophoblasts by increasing polyamines production. These results suggest that the l-arginine-ornithine-polyamine and l-arginine-nitric oxide pathways play a role in placental growth and development.

A case of gain-of-function mutation in calcium-sensing receptor: supplemental hydration is required for renal protection.

AIMS: The calcium-sensing receptor (CaSR) regulates the extracellular calcium level, mainly by controlling parathyroid hormon secretion and renal calcium reabsorption. In gain-of-function CaSR mutations, the genetic abnormalities increase CaSR activity leading to the development of such clinical manifestations as hypercalciuric hypocalcemia and hypoparathyroidism. We report a Japanese case of CaSR gain-of-function mutation and represent a therapeutic intervention based on the functional characteristics of CaSR in renal tubule. METHODS AND RESULTS (CASE): DNA sequence analysis revealed a heterozygous G to T mutation identified in a 12-year-old Japanese girl presenting with sporadic onset of hypercalciuric hypocalcemia and hypoparathyroidism. The mutation is located in the N-terminal extracellular domain of the CaSR gene, one of the most important parts for the three-dimensional construction of the receptor, resulting in the substitution of phenylalanine for cysteine at amino acid 131 (C131F) in exon 3. Based on the diagnosis of the gain-of-function mutation in the CaSR, oral hydrochlorothiazide administration and supplemental hydration were started in addition to calcium supplementation. The combination therapy of thiazide and supplemental hydration markedly reduced both renal calcium excretion and urinary calcium concentration from 0.4-0.7 to less than 0.1 mg/mg (urinary calcium/creatinine ratio) and from 10-15 to 3-5 mg/dl (urinary calcium concentration), respectively. This therapy stopped the progression of renal calcification during the follow-up period. CONCLUSION: Supplemental hydration should be considered essential for the following reasons: (1) calcium supplementation activates the CaSR in the kidney and suppresses renal urinary concentrating ability, (2) the thiazide has a diuretic effect, (3) as calcium supplementation increases renal calcium excretion, the supplemental hydration decreases urinary calcium concentration by increasing urinary volume, thereby diminishing the risk of intratubular crystallization of calcium ion.

Lactational amenorrhea in monkeys: effects of suckling on prolactin secretion.

To determine the acute and chronic effects of suckling on maternal PRL secretion in monkeys, five mother-infant pairs were studied longitudinally on days 40, 80, 120, and 10 after weaning (day 160). Mothers were chronically cannulated and, during blood collections, wore protective nylon vests with mobile tethers. Studies were undertaken during the day and night with the mother and infant undisturbed, during the daytime, before and after the removal of the infant, and during the day and night before and after the reunion of mother and infant. Maternal PRL levels were significantly (P less than 0.05) higher at night than during the day in undisturbed mother-infant pairs. This nocturnal elevation was probably induced by a more intensive interaction of the mother and infant at night than during the day. Basal PRL concentrations in samples collected during these undisturbed settings significantly (P less than 0.05) declined as the postpartum interval continued. The removal of the infant did not perturb maternal PRL patterns. Typically, after reunion of mother and infant, maternal PRL levels were increased significantly (P less than 0.05), reaching maximal levels approximately 2 h after reunion. If PRL secretion, induced by the suckling stimulus, is instrumental in sustaining puerperal infertility, then the increased secretion of PRL that occurs at night during the protracted interval of intense mother-infant interaction may be of particular significance in inhibition of the hypothalamic-pituitary-ovarian axis.

Seasonal changes of plasma estradiol and progesterone in the Japanese monkey (Macaca fuscata fuscata).

The plasma levels of estradiol and progesterone in 5 mature female Japanese monkeys (Macaca fuscata fuscata) throughout a breeding and nonbreeding season were measured by radioimmunoassay. As the breeding season approached, plasma estradiol levels started to increase and a few elevations were detected. However, neither a plasma progesterone rise nor menstrual bleeding was seen. Menstrual bleeding began in December and recurred regularly 4 or 5 times at 4 week intervals until April. During this period, cyclic estradiol elevations (150-250 pg/ml) and luteal progesterone rises (2.0-5.3 ng/ml) were observed. After the last menstruation in March or April, two animals showed an estradiol elevation, but progesterone levels remained in the basal range and amenorrhea persisted. These data indicated that female Japanese monkeys ovulate 4 or 5 times a year, i.e., their breeding season is definitely defined. The changes of both steroids in transitional periods between nonbreeding and breeding, and breeding and nonbreeding seasons, suggested that some follicle development occurred in these periods. The elucidation of the mechanisms involved in the seasonal variation of reproductive functions in the Japanese monkey may provide useful information concerning some aspects of amenorrhea in human females.

Identification of heme oxygenase in human endometrium.

The aim of the present study was to investigate the presence of heme oxygenase (HO)-1 and HO-2 in human endometrium at various stages of the menstrual cycle using RT-PCR, Western blotting, and immunohistochemistry. RT-PCR detected mRNA for HO-1 and HO-2 in human endometrium at all stages of the menstrual cycle. Western blotting also revealed the expression of the two distinct HO proteins throughout the menstrual cycle. HO-1 was constitutively expressed, whereas HO-2 expression was apparently greater in the secretory phase than in the menstrual and proliferative phases. Immunohistochemistry showed that distribution of the two HO isoforms had distinct topographic patterns: HO-1 was observed in endometrial epithelial cells and macrophages, whereas HO-2 was found in endothelial cells and smooth muscle cells of blood vessels in the endometrium. The detection of mRNA and protein for HO-1 and HO-2 in normal human endometrium suggests that the carbon monoxide/HO system may play a role in the local control of endometrial function.

Efficacy of ipriflavone in preventing adverse effects of leuprolide.

The purpose of this study was to evaluate the efficacy of ipriflavone in preventing bone loss, decreasing in serum cholesterol and decreasing the rate of appearance of vasomotor symptoms, as well as the effects of ipriflavone on reduction of myoma volume by estrogen deficiency during treatment with the GnRH analog leuprolide. One hundred two women (mean age, 44.3 +/- 0.53 yr) receiving leuprolide therapy for uterine leiomyoma were randomly allocated to two groups (group A, leuprolide only; group B, leuprolide with ipriflavone). Bone mineral density of the lumbar spine was measured by dual-energy x-ray absorptiometry before and after treatment for 6 months. Levels of serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-C) were measured before treatment and after 3 and 6 months of treatment. Subjects were asked to report the appearance of vasomotor symptoms throughout treatment. Myoma node volumes were measured before treatment and after treatment for 6 months. Bone mineral density was reduced in both groups, with reduction rates of -5.26% in group A and -3.70% in group B (P < 0.01 vs. group A). Changes in bone markers were not significant in either group. TC was significantly increased in both groups, and TG levels were increased significantly after 3 and 6 months of treatment in group A but not in group B. There was no significant difference between these two groups in amount of increase of either TC or TG. LDL-C levels were increased significantly after 3 and 6 months of treatment in both groups, and the differences between the groups (11.7% in group A vs. 7.5% in group B at 3 month and 22.6% in group A vs. 8.4% in group B at 6 month) were significant. Severe vasomotor symptoms were reduced in group B. The rates of reduction of myoma volume were 49.8% in group A and 52.9% in group B; this difference between groups was not significant. Ipriflavone efficaciously alleviated the adverse effects of estrogen deficiency such as bone loss and increase in LDL-C level, and the ability of leuprolide therapy to reduce myoma volume was not decreased by ipriflavone administration.

Synthesis and release of endothelin-1 by human decidual cells.

We have studied whether a novel vasoconstrictor, endothelin-1 (ET-1), is synthesized by and released from human decidual cells in early pregnancy, and whether ET-1 acts directly on their own cells. It was observed that ET-1-like immunoreactivity (ET-1-LI) was released from cultured decidual, but not villous, cells, as a function of time. Reverse-phase high-pressure liquid chromatography of the conditioned media from the decidual cells revealed a major peak of ET-1-LI coeluting with standard ET-1. Phorbol myristate acetate, a protein kinase C activator, dose-dependently increased the release of ET-1-LI from the decidual cells, while a protein kinase C inhibitor, H7, significantly attenuated the stimulatory effect of phorbol myristate acetate on ET-1-LI release. Northern blot analysis demonstrated the expression of messenger RNA for prepro-ET-1 in the decidual tissue, but no such messenger RNA was observed in the villous tissue. The human decidual tissue contained a noninteracting, single class of binding sites demonstrating higher affinity for ET-1 and ET-2 than ET-3. This would be most consistent with the ETA receptor subtype. An ET-1-induced, dose-dependent accumulation of total inositol phosphates was also observed in human decidual cells prelabeled with myo-[3H]inositol. The present results demonstrate for the first time that human decidual cells in early pregnancy can synthesize and release ET-1. These cells also possess specific functional receptors for ET-1 which are coupled to phosphoinositide hydrolysis. Thus our data suggest a possible role for ET-1 in autocrine and/or paracrine function in human decidual cells.

High ratios of free to total insulin-like growth factor-I in early infancy.

Insulin-like growth factor-I (IGF-I) is a major effector of somatic growth and metabolism. In normal, nonpregnant plasma, most of the IGF-I is complexed to specific IGF-binding proteins (IGFBPs), particularly IGFBP-3; only a minor fraction of plasma IGF-I exists in a free form. Recently, we have reported that free IGF-I levels, as measured using a new immunoradiometric assay, are relatively high in maternal plasma during pregnancy because of increased IGFBP-3 proteolytic activity. These high free IGF-I levels are physiologically important for the growth of maternal tissues such as uterus and placenta, which are related to the fetal growth. Growth during early infancy may be a continuation of fetal growth. In the present study, we have analyzed free and total plasma IGF-I and IGFBP-3 proteolytic activity in early infancy. Although the levels of free and total IGF-I were not significantly different in early infancy as compared with prepubertal periods, the ratio of free to total IGF-I (mean = 2.04%) was relatively increased and was similar to the ratio in pregnancy plasma (1.86%). However, unlike in maternal plasma, the high ratios were not totally caused by increased IGFBP-3 proteolytic activity. Our results suggest that there may be an increased conversion of plasma IGF-I to a free form in early infancy. The resultant increase in IGF-I bioavailability could contribute to the rapid somatic growth in early infancy.

The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro.

The effects of endothelin-1 (ET-1) on luteinized human granulosa cells (L-HGCs) have not been examined. It is well known that there are differences of actions of several autocrine/paracrine regulators between L-HGCs and GCs of other species, and therefore the present study was designed to examine the effects of ET-1 1) on intracellular Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-responsive fluorescent indicator Fura-2, 2) on cell proliferation by the nonradioactive method using bromodeoxyuridine, and 3) on basal and gonadotropin-stimulated steroidogenesis, and to examine the expression of ET receptor messenger RNA (mRNA) using freshly isolated and cultured L-HGCs obtained from patients undergoing in vitro fertilization. ET-1 increased [Ca2+]i in L-HGCs in a dose-dependent manner between 1 and 1000 nmol/L. High concentrations (100-1000 nmol/L) of ET-1 produced a more rapid and transient increase in [Ca2+]i than that observed with low concentrations (1-10 nmol/L) of ET-1. The increase in [Ca2+]i elicited by ET-3 (1000 nmol/L) and IRL-1620 (1000 nmol/L), a selective ETB receptor agonist, was 16% and 3% (vs. ET-1, 100%), respectively. BQ-123 (1000 nmol/L), an ETA receptor antagonist, inhibited the increase in [Ca2+]i elicited by ET-1 (by 50% at 1000 nmol/L ET-1 and by > 90% at < 500 nmol/L ET-1). mRNAs for the two known receptor subtypes (ETA and ETB) were also present in L-HGCs; however, the expression of ETA receptor mRNA was much greater than that of ETB receptors. ET-1 stimulated cell proliferation in L-HGCs in a dose-dependent manner (1000 nmol/L, 210.5 +/- 13.1%; 100 nmol/L, 198 +/- 11%; 10 nmol/L, 146 +/- 18%; and 1 nmol/L, 103 +/- 9%; vs. control, 100%). These stimulatory effects were completely blocked by BQ-123 (1000 nmol/L). ET-3 and IRL-1620 had no effects on cell proliferation in L-HGCs. Significant stimulatory effects on cell proliferation by the calcium ionophore, ionomycin (10-1000 nmol/L), were observed. ET-1, ET-3, and IRL-1620 attenuated basal progesterone secretion in L-HGCs. These results suggest that ETA receptor predominantly exist in L-HGCs and that ET-1 may stimulate cell proliferation of L-HGCs by increasing [Ca2+]i via ETA receptors.

A human cDNA encoding the 110-kDa A subunit of RNA polymerase II transcription factor elongin.

A full-length cDNA encoding a human homolog of the approx. 110-kDa subunit (elongin A; El A) of the RNA polymerase II transcription factor, elongin, was isolated and sequenced. Comparison of the open reading frames of the human el A cDNA and the previously characterized rat El A cDNA [Aso et al., Science 269 (1995) 1439-1443] indicates that they are 84% conserved in nucleotide sequence and encode 84% identical proteins.

CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s).

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T-helper- (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti-FcepsilonR1 antibody, cord blood-derived mast cells secreted factor(s) that induced Ca(2+) mobilization in CRTH2-expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell-mediated immune responses such as allergic reactions.

Expression of IL-6 receptor and GP130 in mouse bone marrow cells during osteoclast differentiation.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.