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Short-term storage is valuable method to reuse manipulated embryos.The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h).The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25C.Supplementation of AFP type III (1.0 microgram per mL) improved the developmental competence of embryos stored at 25C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos.Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.

Background Acute pancreatitis (AP) occurs frequently in dogs, but most previous studies examining the diagnosis of AP have used data from secondary care hospitals. Hypothesis/Objectives The aim of this study was to investigate the clinical utility of diagnostic laboratory tests in dogs with AP in a primary care hospital. Animals Sixty-four dogs with clinical signs suggestive of AP diagnosed with nonpancreatic disease (NP) or AP. Methods Medical records were retrospectively reviewed, including diagnostic laboratory tests considered potentially useful in the diagnosis of AP. The diagnostic accuracy of amylase and FUJI DRI-CHEM lipase (FDC lip) were investigated using receiver operating characteristics (ROC). In addition, we verified whether diagnostic laboratory tests were useful for evaluating duration of hospitalization and as biomarkers for monitoring recovery. Results Activities of amylase and FDC lip were significantly higher in the AP group than in the NP group (P = .001, P < .001, respectively). The sensitivity of FDP lip activity for diagnosing AP was 100% (95% confidence interval [CI], 87.7–100%); the specificity was 89.5% (95% CI, 66.9–98.7%). Area under the ROC curve for FDC lip activity was 0.98 (95% CI, 0.93–1). High alanine aminotransferase (ALT) activity was associated with extended duration of hospitalization (P = .04). A significant difference in C-reactive protein (CRP) concentration before and 5 days after treatment was found (P = .001). Conclusions and clinical importance Measurement of FDC lip activity appears useful for diagnosing AP. High ALT activity might be associated with prolonged duration of hospitalization, and CRP might be useful as a biomarker for monitoring recovery from AP.

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.

Bisphosphonates are emerging as an important treatment for osteoporosis. But whether the reduced fracture risk associated with bisphosphonate treatment is due to increased bone mass, improved trabecular architecture and/or increased secondary mineralization of the calcified matrix remains unclear. We examined the effects of bisphosphonates on both the trabecular architecture and matrix properties of canine trabecular bone. Thirty-six beagles were divided into a control group and two treatment groups, one receiving risedronate and the other alendronate at 5-6 times the clinical dose for osteoporosis treatment. After one year, the dogs were killed, and samples from the first lumbar vertebrae were examined using a combination of micro-computed tomography, finite element modeling, and mechanical testing. By combining these methods, we examined the treatment effects on the calcified matrix and trabecular architecture independently. Conventional histomorphometry and microdamage data were obtained from the second and third lumbar vertebrae of the same dogs [Bone 28 (2001) 524]. Bisphosphonate treatment resulted in an increased apparent Young's modulus, decreased bone turnover, increased calcified matrix density, and increased microdamage. We could not detect any change in the effective Young's modulus of the calcified matrix in the bisphosphonate treated groups. The observed increase in apparent Young's modulus was due to increased bone mass and altered trabecular architecture rather than changes in the calcified matrix modulus. We hypothesize that the expected increase in the Young's modulus of the calcified matrix due to the increased calcified matrix density was counteracted by the accumulation of microdamage.

We purified ghrelin from stomach extracts of a teleost fish, the Japanese eel (Anguilla japonica) and found that it contained an amide structure at the C-terminal end. Two molecular forms of ghrelin with 21 amino acids were identified by cDNA and mass spectrometric analyses: eel ghrelin-21, GSS(O-n-octanoyl)FLSPSQRPQGKDKKPP RV-amide and eel ghrelin-21-C10, GSS(O-n-decanoyl) FLSPSQRPQGKDKKPPRV-amide. Northern blot and RT-PCR analyses revealed high gene expression in the stomach. Low levels of expression were found only in the brain, intestines, kidney and head kidney by RT-PCR analysis. Eel ghrelin-21 increased plasma growth hormone (GH) concentrations in rats after intravenous injection; the potency was similar to that of rat ghrelin. We also examined the effect of eel ghrelin on the secretion of GH and prolactin (PRL) from organ-cultured tilapia pituitary. Eel ghrelin-21 at a dose of 0.1 nM stimulated the release of GH and PRL, indicating that ghrelin acts directly on the pituitary. The present study revealed that ghrelin is present in fish stomach and has the ability to stimulate the secretion of GH from fish pituitary. A novel regulatory pathway of GH secretion by gastric ghrelin seems to be conserved from fish to human.

We recently demonstrated that suppressed bone remodeling allows microdamage to accumulate and causes reductions in some mechanical properties. However, in our previous study, 1 year treatment with high-dose etidronate (EHDP) did not increase microdamage accumulation in most skeletal sites of dogs in spite of complete remodeling suppression and the occurrence of spontaneous fractures of ribs and/or thoracic spinous processes. This study evaluates the effects of EHDP on microdamage accumulation and biomechanical properties before fractures occur. Thirty-six female beagles, 1-2 years old, were treated daily for 7 months with subcutaneous injections of saline vehicle (CNT) or EHDP at 0.5 (E-low) or 5 mg/kg per day (E-high). After killing, bone mineral measurement, histomorphometry, microdamage analysis, and biomechanical testing were performed. EHDP treatment suppressed intracortical and trabecular remodeling by 60%-75% at the lower dose, and by 100% at the higher dose. Osteoid accumulation caused by a mineralization deficit occurred only in the E-high group, and this led to a reduction of mineralized bone mass. Microdamage accumulation increased significantly by two- to fivefold in the rib, lumbar vertebra, ilium, and thoracic spinous process in E-low, and by twofold in the lumbar vertebra and ilium in E-high. However, no significant increase in damage accumulation was observed in ribs or thoracic spinous processes in E-high where fractures occur following 12 months of treatment. Mechanical properties of lumbar vertebrae and thoracic spinous processes were reduced significantly in both E-low and E-high. These findings suggest that suppression of bone remodeling by EHDP allows microdamage accumulation, but that osteoid accumulation reduces production of microdamage.

Background. Atherosclerosis is the main lesion in allografts undergoing chronic rejection. We investigated the effect of OP-2507 (prostaglandin I2 analogue) and OKY-046 (thromboxane A2 synthetase inhibitor) on graft atherosclerosis morphologically and the production of eicosanoids in grafts in a rat aortic allograft model. Methods. Abdominal aortic allografts of Lewis (RT-1l) rats were transplanted orthotopically into fully major histocompatibility complex mismatched Wistar King A/Qdj (RT-1u) rats that were subcutaneously administered OP-2507 (0.1 mg/kg/d) or OKY-046 (125 mg/kg/d), or both, with an osmotic pump. Four, 8, or 12 weeks later, the grafts were harvested and examined histologically, and the concentration of eicosanoids in the grafts were analyzed. Results. Lewis aortic allografts in Wistar King A recipients with no treatment displayed atherosclerosis, which involved gradual intimal thickening and medial thinning with continuous inflammation in adventitia. Neither OP-2507 nor OKY-046 treatment affected the intensity of adventitial inflammation. Although inhibition of medial thinning or a decrease in medial nuclear density was not observed, OKY-046 administration alone significantly inhibited an increase in intimal thickness. OP-2507 administration alone significantly inhibited a decrease in medial nuclear density and intimal thickening. Combined treatment with OP-2507 and OKY-046 further decreased the alteration of media and intima. The ratio of thromboxane B2 and 6-keto-prostaglandin F1α in the grafts was significantly reduced by OKY-046 but not by OP-2507 alone. Conclusions. We have demonstrated that atherosclerosis in aortic allografts is inhibited by the continuous administration of either OP-2507 or OKY-046, and a combination of both agents strongly increases this inhibitory effect. Amelioration of balance in eicosanoid production in the grafts by the use of thromboxane A2 synthetase inhibitor and the simultaneous usage of stable prostaglandin I2 analogue may be a strategy for preventing atherosclerosis that results from chronic rejection. (Surgery 2001;129:595-605.)

Suicide gene therapy using ganciclovir (GCV) with transfection of the herpes thymidine kinase (HSVtk) gene has been studied for cancer therapy. The present study demonstrates an efficient method of suicide gene therapy for multiple hepatic tumors, involving repetitive transfection of the HSVtk gene driven by the alpha-fetoprotein (AFP) promoter using hemagglutinating virus of Japan (HVJ)-liposomes. AFP-producing cells (HUH7) and AFP-nonproducing cells (LS180) were injected subcutaneously (s.c.) to establish tumors in nude mice. Two plasmid constructs, bacterial LacZ gene driven by the AFP promoter (AFPLacZ), and HSVtk gene driven by the AFP promoter (AFPTK1) were encapsulated into the HVJ-liposome and used. When AFPLacZ was injected into the s.c. tumors, expression of LacZ gene was confined to HUH7 tumors. Repeated transfection of AFPTK1 followed by GCV treatment markedly suppressed growth of HUH7 tumors, and apoptosis of HUH7 cells was recognized in the tumor. Next, HUH7 cells were injected into the portal vein in severe combined immunodeficiency mice to establish a hepatic tumor model. After inoculation with the tumor, HVJ-liposomes containing the AFPTK1 plasmid vector were injected into the portal vein via the splenic hilum, followed by GCV treatment. This gene therapy significantly inhibited the growth of tumors in the liver and markedly improved survival. Three injections of the AFPTK1 plasmid vector completely inhibited tumor growth. This procedure seems to have great potential for the treatment of multiple hepatic tumors.

We performed animal experiments to test the hypothesis that active oxygen species (AOS) play a major role in adjuvant-induced arthritis in rats and to determine whether large-dose ascorbic acid administration would suppress the development of arthritis, reducing the level of damaging AOS in the same animal model. Arthritis was induced in male Lewis rats by adjuvant injection into the base of the tail. Ascorbic acid at doses of 0.5, 1.0, and 2.0 g/kg body weight (BW) was injected intraperitoneally twice each week for 3 weeks (9 rats per group). The BW, hind paw edema, and arthritis score of the extremities were monitored during the period. On day 21, synovial tissues obtained from the ankle joints were examined histologically and for the activity of superoxide dismutase (SOD). The SOD activity in the red blood cells (RBC) was also measured. The arthritic control rats showed significant increases in paw volume and arthritis score from day 11. These changes were dose-dependently reduced by ascorbic acid administration. The infiltration of inflammatory cells into the synovial tissues was markedly decreased by ascorbic acid. The increases in SOD activities produced by the adjuvant injection were significantly reduced in both the synovium and the RBC at ascorbic acid doses of 1.0 and 2.0 g/kg BW. In conclusion, large-dose ascorbic acid administration reduced the increases in hind paw inflammatory edema, arthritis in the extremities, and infiltration of the inflammatory cells into the synovial tissue in the adjuvant-induced arthritis rats. Since these anti-arthritic effects were associated with a decrease in SOD activities in both the synovium and RBC, the decrease in SOD activity could be one of the mechanisms underlying the suppressive effects of large-dose ascorbic acid on the development of arthritis in this animal model, inhibiting the damaging AOS.

Intermittent administration of parathyroid hormone (PTH) has an anabolic effect in cancellous bone of osteoporotic humans. However, the effect of PTH on cortical bone with Haversian remodeling remains controversial. The aim of this study was to determine the effects of biosynthetic human PTH(1-34) on the histology and mechanical properties of cortical bone in rabbits, which exhibit Haversian remodeling. Mature New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 micrograms/kg/day or 40 micrograms/kg/day for 140 days. Body weight in rabbits treated with PTH did not change significantly over the experimental period. Serum calcium and phosphate were within the normal range, but a 1 mg/ml increase in serum calcium was observed in rabbits given the higher dose of PTH. Histomorphometry of cortical bone in the midshaft of the tibia showed significant increases in periosteal and endocortical bone formation in these rabbits. Intracortical bone remodeling in the tibia was activated and cortical porosity increased by PTH. Cross-sectional bone area and bone mass of the midshaft of the femur increased significantly after PTH treatment. Ultimate force, stiffness, and work to failure of the midshaft of the femur of rabbits given the 40 micrograms dose of PTH were significantly greater than those in the control group, whereas elastic modulus was significantly lower than that in the rabbits given the 10 micrograms dose of PTH, but not different from controls. In the third lumbar vertebra, PTH increased both formation and resorption without increasing cancellous bone volume. The increases in bone turnover and cortical porosity were accompanied by concurrent increases in bone at the periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the ultimate stress, stiffness, and work to failure of the femur.

A radio frequency identification (RFID) transponder covering the 13.56 MHz band was adapted to minimize its volume so that it could be placed in the pulp chamber of an endodontically treated human tooth. The minimized transponder had a maximum communication distance of 30 mm. In an animal experiment, the transponder was fixed in the cavity of a mandibular canine of a dog. An RFID reader positioned close to the dog's face could communicate with the transponder in the dog's tooth. In certain cases, the system is applicable for the personal identification procedures for hospitalized patients instead of an identification wristband.

Bone marrow stromal cell Ag-1 (BST-1; CD157)-deficient mice were generated to examine the immunologic roles of the molecule in vivo. In BST-1−/− mice, the development of peritoneal B-1 cells was delayed, and CD38low/− B-lineage cells were increased in the bone marrow and spleen. Partial impairment of thymus-independent (TI-2) and thymus-dependent (TD) Ag-specific immune responses was noted in the systemic and mucosal compartments of BST-1−/− mice, respectively. Although serum Ig levels as well as TD and TI-1 Ag-specific systemic immune responses were normal, the TI-2 Ag-induced IgG3 response was selectively impaired. Oral immunization of BST-1−/− mice with cholera toxin, a potent TD Ag for the induction of IgA response, resulted in the poor production of Ag-specific Abs at the intestinal mucosa accompanied by the reduced number of Ag-specific IgA-producing cells in the lamina propria. These results indicate that BST-1 has roles in B cell development and Ab production in vivo.

Cordyceps species, which are used in Chinese traditional medicines, are fungal parasites of insects. In this study the partial nucleotide sequences of 18 S ribosomal DNA from four Cordyceps species were determined and compared with the sequences of published ascomycetes. The sequence data support the concept that Cordyceps species belong to the pyrenomycetes. Based on sequence data the phylogenetic tree was constructed using the neighbor-joining (NJ) method. Diversity in the phylogenetic tree was found for Cordyceps species. A new classification of Cordyceps species can be constructed based on the phylogenetic information obtained from such rDNA sequences.

Background. In the present study we attempted to evaluate the role of the graft as an alloantigen in induction and maintenance of donor-specific prolongation of allograft survival by short course(s) of FK506 treatment in rat heart transplantation. Materials and methods. In the WKA/Qdj-to-LEW combination group, FK506 (3 mg/kg/day, 7 days, sc) was administered from Day 0 after cardiac transplantation. A second WKA/Qdj cardiac graft was transplanted on Day 14 into the LEW recipient after removal of the first graft on Day 3 or Day 7. In other groups an additional course of FK506 (3 mg/kg/day, 7 days, sc) was given from Day 35. A second WKA/Qdj cardiac graft was transplanted on Day 49 into the LEW recipient after removal of the first graft on Day 7 or Day 28. Results. A short course of FK506 treatment allowed for survival prolongation of a cardiac allograft [mean survival time (MST) = 42.8 days]. Removal of the first graft on Day 7 (MST = 16.4 days) but not on Day 3 (MST = 10.2 days) caused donor-specific prolongation of the second allograft survival, which was significantly shorter than that in the recipient without the graftectomy (MST = 32.8 days). When graftectomy was performed on Day 3, there were immunohistochemically detectable levels of donor class II expressing cells in the recipient spleen on Day 7, indicating that the presence of the graft more effectively induced unresponsiveness than microchimerism alone. The additional course of FK506 treatment on Day 35 maintained graft survival (MST > 90.6 days). Removal of the first graft on Day 7 (MST = 13.5 days) or Day 28 (MST = 24.7 days) did not show significant prolongation of the second allograft survival, whereas significant survival prolongation of the second graft was observed in the recipient without the graftectomy (MST = 38.8 days). However, marked survival prolongation of the second donor-specific allograft in half of the recipients that had received the second course of FK506 treatment after graftectomy on Day 28 indicates that residual donor antigen can function as tolerizing antigen with an FK506 supplement. Conclusions. We concluded that the persistence of a graft more effectively induces and maintains donor-specific unresponsiveness than does the chimeric state of graft-derived cells alone under immunosuppression by FK506 in the rat heart transplantation model.

This study was conducted to examine the effect of gamma-interferon (IFN-gamma) on experimental metastasis formation by murine colon 26 adenocarcinoma in BALB/c mice. We found that the number of experimental lung metastases was increased after colon 26 cells were pretreated for 1 h with as little as 1 OIU/ml of IFN-gamma. 5-[125I] iodo-2'-deoxyuridine-radiolabeled colon 26 cells pretreated with IFN-gamma remained at higher level in the lung at 24h after intravenous injection than when the cells were not pretreated. In vivo elimination of asialo GM1-positive cells increased the number of lung metastases and, in such mice, there was no longer a difference in metastatic ability between control and IFN-gamma-treated cells. Colon 26 cells were completely resistant to lysis by isolated splenocytes. Splenocytes incubated in vitro with interleukin 2 exhibited moderate cytotoxicity against colon 26 cells, but there were no significant differences between control and IFN-gamma-treated cells. Colon 26 cells pretreated with IFN-gamma demonstrated resistance to tumor necrosis factor alpha-mediated growth inhibition. The enhancement of metastases by IFN-gamma was dependent on de novo protein synthesis since the enhancement was abolished by cycloheximide. Taken together, the data suggest that the metastatic ability of colon 26 cells pretreated with IFN-gamma is significantly higher due to the resistance to asialo GM1-positive cells accompanied with de novo protein synthesis.

Hyaluronic acid (HA), which is known to play an important role in wound healing, was incorporated in an artificial skin material and studied for its potential to create a wound bed which would support a skin graft. Collagen sponge based artificial skin was soaked in 0.3% HA in phosphate buffered saline and grafted onto skin defects in rats. Control grafts were soaked in normal saline solution. HA incorporated implants and control implants were simultaneously grafted onto wounds made on either side of the spine. To examine the effect of HA incorporation, the percentage area of cellular tuft infiltration and the number of capillaries present in the graft matrix were evaluated at 7 and 14 days after the operation. At postoperative day 7, there was a statistically significant difference in the number of capillaries in the matrix of the experimental versus the control implants. There was no difference in the percentage area of cellular tuft infiltration. At postoperative day 14, all implants exhibited better ingrowth of granulation tissue than at day 7. The differences between the experimental and control implants were statistically significant with respect to both the percentage area of cellular tuft infiltration and the number of capillaries. It is therefore concluded that in artificial skin HA incorporation accelerates the ingrowth of granulation tissue, making a more suitable graft bed.

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause Marfan syndrome (MFS). Frequent observation of cattle with a normal withers height, but lower body weight than age-matched normal cattle, was recently reported among cattle sired by phenotypically normal Bull A, in Japanese Black cattle. These cattle also showed other characteristic features similar to the clinical phenotype of human MFS, such as a long phalanx proximalis, oval face and crystalline lens cloudiness. We first screened a paternal half-sib family comprising 36 affected and 10 normal offspring of Bull A using the BovineSNP50 BeadChip (illumina). Twenty-two microsatellite markers mapped to a significant region on BTA10 were subsequently genotyped on the family. The bovine Marfan syndrome-like disease (MFSL) was mapped onto BTA10. As FBN1 is located in the significant region, FBN1 was sequenced in Bull A, and three affected and one normal cattle. A GA mutation at the intron64 splicing accepter site (c.8227-1GA) was detected in 31 of 36 affected animals (84.7%). The c.8227-1GA polymorphism was not found in 20 normal offspring of Bull A or in 93 normal cattle unrelated to Bull A. The mutation caused a 1-base shift of the intron64 splicing accepter site to the 3' direction, and a 1-base deletion in processed mRNA. This 1-base deletion creates a premature termination codon, and a 125-amino acid shorter Fibrillin-1 protein is produced from the mutant mRNA. We therefore conclude that the c.8227-1GA mutation is causative for MFSL. Furthermore, it was suggested that Bull A exhibited germline mosaicism for the mutation, and that the frequency of the mutant sperm was 14.9%.

When rats were given a supramaximal dose of caerulein (infused intravenously at 5 μg/kg.h for 4 h) they developed acute pancreatitis characterized by significantly raised amylase levels in the blood. In this model of acute pancreatitis, reduced gastric adenylate energy charge levels were observed, and the leakage of the lysosomal enzyme, cathepsin B, from gastric lysosomes and of the mitochondrial enzyme, malate dehydrogenase, from gastric mitochondria were both significantly accelerated compared with the control group. The intragastric administration of the anti-ulcer agent, teprenone, at a dose of 5 mg/kg (twice before caerulein infusion) significantly inhibited this gastric damage accompanying acute pancreatitis. These results suggest that gastric subcellular organelle fragility may play an important role in the pathogenesis of impaired gastric energy metabolism accompanying acute pancreatitis, and indicate the possible usefulness of teprenone in preventing this gastric damage.

Necrosis of the femoral head and osteopenia were examined histopathologically in stroke-prone spontaneously hypertensive rats (SHRSPs) aged 6 to 36 weeks and compared with that of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs). Avascular necrosis of the femoral head was frequently observed, mainly in the young SHRSPs and SHRs (about 8 to 15 weeks of age). SHRSPs had the highest incidence of femoral head necrosis among the three strains. This necrotic change in the femoral head was considered to be secondary ischemia induced by angiospasm or arteriosclerosis, similar to the disorders observed in the brain, kidney, and heart in SHRSPs. However, the complication occurred in spite of treatment with antihypertensive agents (ACE inhibitor: enalapril, spirapril) even though other ischemic disorders such as brain hemorrhage and renal infarction were prevented, indicating that the femoral head necrosis in SHRSPs was not due to hypertensive complications induced by angiospasm or arteriosclerosis. Bone mineral density (BMD) of the femoral bone was significantly lower in SHRSPs, and the femoral heads in this strain were the most easily deformed by loads applied during compression tests. Histopathologically, the infarctions were encountered on the lateral side of the epiphysis, but no thrombi were observed. The lateral side of the epiphysis is the anatomic site where the weight load is greatest and the site where the nutritive artery enters. Our results strongly suggest that the coexistence of vulnerable bone matrix and physical weight load to the nutritive artery plays a crucial role in the occurrence of femoral head necrosis in SHRSPs, whether based on generalized or localized osteopenia.

Long-term ethanol feeding has been shown to selectively reduce hepatic mitochondrial glutathione content by impairing mitochondrial uptake of this thiol. In this study, we assessed the role of this defect in evolution of alcoholic liver disease by examining the mitochondrial glutathione pool and lipid peroxidation during progression of experimental alcoholic liver disease to centrilobular liver necrosis and fibrosis. Male Wistar rats were intragastrically infused with a high-fat diet plus ethanol for 3, 6 or 16 wk (the duration that resulted in induction of liver steatosis, necrosis and fibrosis, respectively). During this feeding period, the cytosolic pool of glutathione remained unchanged in the ethanol-fed animals compared with that in pair-fed controls. In contrast, the mitochondrial pool of glutathione selectively and progressively decreased in rats infused with ethanol for 3, 6 or 16 wk, by 39%, 61% and 85%, respectively. Renal mitochondrial glutathione level remained unaffected throughout the experiment. Serum ALT levels increased significantly in the ethanol-fed rats at 6 wk and remained elevated at 16 wk. In the mitochondria with severely depleted glutathione levels at 16 wk, enhanced lipid peroxidation was evidenced by increased malondialdehyde levels. Thus a progressive and selective depletion of mitochondrial glutathione is demonstrated in the liver in this experimental model of alcoholic liver disease and associated with mitochondrial lipid peroxidation and progression of liver damage.

1. Studies have been performed to determine the effect of intraduodenal food on pancreatic secretion of lysosomal enzymes. 2. Intraduodenal instillation of a liquid meal (3 g/kg body weight; 15.3% protein, 19.7% fat, 59.7% carbohydrate) caused significant increases in pancreatic juice volume and pancreatic secretion of amylase and protein compared with basal values for 2 h after instillation in anaesthetized rabbits. 3. Intraduodenal instillation of a liquid meal also caused significant increases in pancreatic secretion of three lysosomal enzymes (cathepsin B, N-β-acetyl-galactosaminidase and N-acetyl-β-glucosaminidase) compared with basal values for 2 h after instillation. 4. In addition, there were significant correlations between cathepsin B secretion and amylase secretion (r = 0.7764, P 5. These results are evidence for the localization of lysosomal enzymes in the secretory granules-zymogen granules in normal acinar cells, and also indicate that the pancreatic secretion of lysosomal enzymes is gut-hormone-regulated.

The study investigated the protective effect of a new synthetic protease inhibitor, E-3123, a 4-guanidinobenzoate methanesulphonate, on the exocrine pancreas in caerulein-induced pancreatitis of rats both in vivo and in vitro. Hyperamylasaemia, pancreatic oedema and congestion of amylase, as well as cathepsin B leakage from lysosomes and malate dehydrogenase leakage from mitochondria, were prevented by infusion of 5 mg/kg.h E-3123 particularly when infused for 2 h before and during 5 μg/kg.h caerulein infusion for 3.5 h. The results indicate that E-3123 plays its protective roles against pancreatitis in the subcellular compartments such as lysosomes and mitochondria, and that such a low molecular weight protease inhibitor as E-3123 may be clinically useful in the treatment of acute pancreatitis.

It has been suggested that acetylcholine (ACh) might play an important role in carotid body chemoreception. However, a unified explanation of the actions of ACh in this organ has been lacking, due in part to the opposite pharmacological effects of this agent on carotid sinus nerve discharge in different animal species, most notably the cat, where ACh is excitatory, versus the rabbit, where it is inhibitory. In the present study, we utilized receptor binding techniques to compare the nicotinic and muscarinic receptor populations in the carotid bodies of these two species. Our results with the nicotinic ligand 125I-alpha-bungarotoxin and the muscarinic ligand 3H-quinuclidinyl benzilate suggest that nicotinic receptors predominate in the cat by a ratio of 2:1, while in the rabbit, a 12:1 ratio favors muscarinic receptors. Our data suggest that the relative numbers of muscarinic and nicotinic receptors in the carotid bodies of these two species determine the excitatory or inhibitory actions of exogenously administered ACh in this chemoreceptor organ.

We studied the effect of short-term (3 h) pancreatic duct obstruction (PDO) on the exocrine pancreas and on the secretion of lysosomal enzymes into the pancreatic juice of rabbits during stimulation by pancreatic secretagogues. The following evaluations were made: serum amylase levels, pancreatic water content, pancreatic amylase, trypsinogen and cathepsin B content, and output of pancreatic enzymes and lysosomal hydrolases when stimulated by secretin and caerulein as well as the distribution of cathepsin B in subcellular fraction. Cellular fragility (LDH leakage from dispersed acini) and subcellular organellar fragility (cathepsin B leakage from lysosomes and malate dehydrogenase leakage from mitochondria) were also evaluated. PDO for 3 h plus secretin infusion caused a significant rise in serum amylase levels, pancreatic water content, and pancreatic amylase and trypsinogen content due to congestion of digestive enzymes during PDO. There was also a redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction and increased cellular and subcellular organellar fragility. In normal rabbits and in those with only secretin infusion, caerulein stimulated the secretion of cathepsin B into pancreatic juice. Just after PDO, the secretion of cathepsin B, amylase and trypsinogen significantly decreased. By 24 h after PDO, the output of cathepsin B stimulated by caerulein and secretin had increased significantly. Amylase and trypsinogen output were also significantly increased at this stage, in both the secretin and caerulein fractions. These results indicate that the secretion of lysosomal enzymes into pancreatic juice is stimulated by gut hormones, such as caerulein, in the normal physiological state and in pathological states, such as PDO. These results also show an important role of increased cellular and subcellular organellar fragility in the pathogenesis of pancreatic injuries induced by PDO and augmented secretion of both lysosomal enzymes and pancreatic digestive enzymes in the recovery stage after PDO and their important roles at this stage. Lysosome enzymes also seem to play some physiological roles in the pancreatic ductal system in normal physiological states as well as their roles in pathological states, because cathepsin B can activate trypsinogen, and trypsin can activate many other enzymes.

Producción Científica, Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [SH]quinuclidinylbenzitate ([3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1/~M atropine) with a K d = 71.46 pM and a Bm~ x = 9.23 pmol/g tissue. These binding parameters and the pharmacology of the displaceable [SH]QNB binding sites are similar to specific muscannic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that musearinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [3H]QNB binding sites to Iobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100/~M) and bethanechol (100 ~tM) were shown to inhibit both the release of catecholamines and the increased CSN activity evoked by nicotine (50/~M) stimulation of the in vitro carotid body, Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.

Lysosomal hydrolases such as cathepsin B are apically secreted from rabbit pancreatic acinar cells via a regulated as opposed to a constitutive pathway. Intravenous infusion of the cholecystokinin analogue caerulein results in highly correlated apical secretion of digestive and lysosomal enzymes, suggesting that they are discharged from the same presecretory compartment (zymogen granules). Lysosomal enzymes appear to enter that compartment as a result of missorting. After 7 h of duct obstruction is relieved, caerulein-stimulated apical secretion of cathepsin B and amylase is increased, but the ratio of cathepsin B to amylase secretion is not different than that following caerulein stimulation of animals never obstructed. These findings indicate that duct obstruction causes an increased amount of both lysosomal and digestive enzymes to accumulate within the secretagogue releasable compartment but that duct obstruction does not increase the degree of lysosomal enzyme missorting into that compartment. Pancreatic duct obstruction causes lysosomal hydrolases to become colocalized with digestive enzymes in organelles that, in size and distribution, resemble zymogen granules but that are not subject to secretion in response to secretagogue stimulation. These organelles may be of importance in the development of pancreatitis.

Ouabain, a cardiac glycoside and inhibitor of Na + , K + -ATPase, is now believed to be a steroid hormone in mammals. We have recently identified ouabain immunoreactivity in the plasma of the tilapia, a euryhaline teleost. Changes in plasma concentrations of immunoreactive ouabain (20–40 pM) in response to salinity change were well correlated with the changes in plasma osmolality and cortisol. Our previous studies have shown that cortisol rapidly inhibits prolactin (PRL) release from the tilapia pituitary by suppressing intracellular Ca 2+ ([Ca 2+ ] i ) and cAMP. In the present study, low doses of ouabain (10–1000 pM) inhibited PRL release dose-dependently during 2–24 h of incubation. There was no effect on growth hormone (GH) release, except for a significant increase at 1000 pM during 8–24 h of incubation. Significant dose-related increases in PRL release were observed at higher doses of ouabain (100–1000 nM), whereas significant inhibition was seen in GH release at 1000 nM during 2–24 h of incubation. Ouabain at 1–100 pM had no effect on Na + , K + -ATPase activity of the pituitary homogenate. The enzyme activity was inhibited by higher concentrations of ouabain, 10% at 1 nM, 15% at 10 nM, 28% at 100 nM, and 45% at 1000 nM. Ouabain also attenuated stimulation of PRL release by the Ca 2+ ionophore, A23187, and by a combination of dibutyryl cAMP and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthin. Intracellular Ca 2+ concentrations were monitored in the dispersed PRL cells with the Ca 2+ -sensitive dye, fura-2. Ouabain at 1 nM reversibly reduced [Ca 2+ ] i within seconds, whereas 1 μM ouabain increased [Ca 2+ ] i . A rapid reduction in [Ca 2+ ] i was also observed when PRL cells were exposed to 1 μM cortisol, whereas there was no consistent effect at 1 nM. These results suggest that ouabain at physiological concentrations rapidly inhibits PRL release from the tilapia pituitary by suppressing intracellular Ca 2+ and cAMP metabolism. The stimulation of PRL release by high concentrations of ouabain (100–1000 nM) may result from an increase in [Ca 2+ ] i , and subsequent depolarization due to the inhibition of Na + , K + -ATPase activity.

There is considerable evidence that the GH/IGF-I axis plays an important role in female reproduction. We report the isolation and characterization of the GH receptor (GH-R) and its gene expression profile during oogenesis in the tilapia, Oreochromis mossambicus. cDNA encoding GH-R was cloned and sequenced from the tilapia liver. The predicted GH-R preprotein consisted of 635 amino acids and contained a putative signal peptide, an extracellular region with a characteristic motif, a single transmembrane region, and a cytoplasmic region with conserved box 1 and 2 domains. The tilapia GH-R shared 34-74% identities with known GH-Rs in vertebrates. A binding assay using COS-7 cells showed that the cloned GH-R bound specifically to tilapia GH. Northern blot analysis showed a single mRNA transcript in the liver and ovary. In situ hybridization revealed intense signals of GH-R in the cytoplasm and nucleus of immature oocytes. The granulosa and theca cells surrounding vitellogenic oocytes also contained the GH-R mRNA signals. About a tenfold greater level of GH-R mRNA was found in the immature oocytes versus the mature oocytes, along with high levels of IGF-I mRNA. There were no significant changes in mRNA levels of GH-R and IGF-I in the liver or in plasma IGF-I levels during oocyte development. No correlation was found between hepatic GH-R mRNA and ovarian GH-R mRNA. These results suggest that the GH/IGF-I axis in the ovary may be involved in the early phases of oogenesis, under a different regulatory mechanism of GH-R gene expression from that of the liver.

Suppression of bone turnover using anti-resorptive agents such as bisphosphonates prevents bone loss but also may increase tissue mineralization. This may make the bone more prone to initiate microcracks. The objective of this study was to determine whether suppression of remodeling caused by treatment of dogs for 1 year with five times the clinical dose of either alendronate or risedronate is associated with increased tissue mineralization and whether it changes the nature of the mineral crystal. Thirty-five dogs were divided into three weight-matched groups and treated daily for 1 year with a subcutaneous injection of saline (CON, n = 12), oral risedronate (RIS, 0.5 mg/kg/day, n = 11), or oral alendronate (ALN, 1.0 mg/kg/day, n = 12). Density fractionation, peripheral quantitative computerized tomography (pQCT), and quantitative backscattered electron microscopy (qBSE) were used to evaluate changes in mineral content of bone tissue from the vertebrae or ribs. Infrared microspectroscopy (IR) and X-ray diffraction were used to assess the quality of the mineral and some aspects of collagen structure in the thoracic vertebrae and iliac crest. Following 12 months of treatment, there was a significant shift toward higher density bone in both ALN (P = 0.04) and RIS (P = 0.002) by density fractionation methods. IR, pQCT, and qBSE did not detect any significant differences in mineralization, probably because of their lower sensitivity and/or because of the smaller region of interest they sample. No significant differences were found in the maturity of the mineral crystals or in their length or size. We conclude that treatment for 1 year with high doses of bisphosphonates which suppress bone remodeling up to 90% increases tissue mineralization, but does not change the nature of the mineral crystal.

Ouabain, a cardiac glycoside and inhibitor of Na(+), K(+)-ATPase, is now believed to be a steroid hormone in mammals, involved in blood pressure and volume regulation and possibly acting as a natriuretic hormone. We have identified ouabain-like immunoreactivity in the plasma and tissues of a euryhaline teleost, the tilapia (Oreochromis mossambicus), by means of solid-phase extraction followed by a specific radioimmunoassay. Plasma concentrations of immunoreactive ouabain were 5-20pg/ml. Ouabain immunoreactivity was detected in all the tissues examined, with highest concentrations in the head kidney followed by intestine and body kidney. When the fish in fresh water were transferred to seawater, plasma osmolality increased significantly after 2, 4, 8, and 24h. Significant increases were observed in plasma ouabain immunoreactivity after 4 and 24h, and a significant correlation was seen between ouabain immunoreactivity and plasma osmolality. There was also a significant correlation between the plasma osmolality and cortisol concentrations. Upon transfer from seawater to fresh water, significant increases were seen in plasma cortisol after 4 and 8h and in immunoreactive ouabain after 4h. When the correlation was analyzed using all the data obtained during the two transfer experiments, plasma ouabain immunoreactivity and cortisol were significantly correlated with plasma osmolality, whereas there was a significant negative correlation between plasma prolactin and osmolality. A significant positive correlation was also seen between plasma cortisol and ouabain immunoreactivity. These results suggest that immunoreactive ouabain may be involved, together with cortisol, in the maintenance of hydromineral balance in the tilapia.

Exposure to cortisol inhibits prolactin (PRL) release from the tilapia pituitary within 10-20min through a plasma membrane-associated, non-genomic pathway. In the present study, in vitro effects of cortisol on the release and mRNA levels of two PRLs (PRL(188) and PRL(177)) and growth hormone (GH) were examined in the organ-cultured pituitary of the Mozambique tilapia, Oreochromis mossambicus. The PRL release was significantly greater in hyposmotic (300mOsmolal) than in hyperosmotic (350mOsmolal) medium during the 2-8h of incubation. The mRNA levels of two PRLs, as estimated by RNase protection assay, were increased after 8h in hyposmotic medium. Cortisol (200nM) inhibited the release of two PRLs under hyposmotic conditions within 1h, and the inhibitory effects lasted for 24h. Cortisol also reduced the gene transcription of both PRLs during 2-8h of incubation but not after 24h. No effect of cortisol was observed on PRL release or on its mRNA levels under hyperosmotic condition. There was no significant effect of medium osmolality on the release or mRNA levels of GH during 8h of incubation. However, GH release was significantly stimulated by cortisol after 4h, and the effect lasted for 24h under both hyposmotic and hyperosmotic conditions. Cortisol also caused a significant increase in GH mRNA levels at 8 and 24h. These results suggest that cortisol inhibits PRL release from the tilapia pituitary through non-genomic and also through transcriptional pathways, while stimulating GH release through classical genomically mediated glucocorticoid actions.

Glucocorticoids are known to impede somatic growth in a wide range of vertebrates. In order to clarify the mechanisms through which they may act in an advanced teleost fish, we examined the effects of cortisol administration on the growth hormone (GH)/insulin-like growth factor-I (IGF-I)/IGF-binding protein (IGFBP) system in the tilapia (Oreochromis mossambicus). In a short-term experiment, fish were injected intraperitoneally with cortisol (2 or 10 microg/g), and killed at 2, 4, 8 and 24 h after the injection. In a longer-term experiment, fish were killed 24 and 48 h after cortisol injection (2, 10 and 50 microg/g). Cortisol at doses of 2 and 10 microg/g significantly increased IGFBPs of four different sizes (24, 28, 30, and 32 kDa) in the plasma within 2 h without altering plasma levels of IGF-I or GH. On the other hand, cortisol at doses of 10 and 50 microg/g significantly reduced plasma IGF-I levels after 24 and 48 h. IGF-I mRNA levels in the liver were also significantly reduced by cortisol at doses of 10 and 50 microg/g after 48 h, suggesting that a decrease in plasma IGF-I levels is mediated through the attenuation of IGF-I gene expression in the liver. In contrast, no significant change was observed in plasma or pituitary contents of GH at any time point examined, which would appear to indicate that cortisol reduces IGF sensitivity to GH (GH-resistance). These results clearly indicate that cortisol induces a rapid increase in plasma IGFBPs and a more delayed decrease in IGF-I production. The dual mode of cortisol action may contribute to the inhibitory influence of cortisol on somatic growth in teleosts.

Interleukin (IL)-6 is a pleiotropic cytokine that not only affects the immune system, but also acts in other biological systems and many physiological events in various organs. In a target cell, IL-6 can simultaneously generate functionally distinct or sometimes contradictory signals through its receptor complex, IL-6Ralpha and gp130. One good illustration is derived from the in vitro observations that IL-6 promotes the growth arrest and differentiation of M1 cells through gp130-mediated STAT3 activation, whereas the Y759/SHP-2-mediated cascade by gp130 stimulation has growth-enhancing effects. The final physiological output can be thought of as a consequence of the orchestration of the diverse signaling pathways generated by a given ligand. This concept, the signal orchestration model, may explain how IL-6 can elicit proinflammatory or anti-inflammatory effects, depending on the in vivo environmental circumstances. Elucidation of the molecular mechanisms underlying this issue is a challenging subject for future research. Intriguingly, recent in vivo studies indicated that the SHP-2-binding site- and YXXQ-mediated pathways through gp130 are not mutually exclusive but affect each other: a mutation at the SHP-2-binding site prolongs STAT3 activation, and a loss of STAT activation by gp130 truncation leads to sustained SHP-2/ERK MAPK phosphorylation. Although IL-6/gp130 signaling is a promising target for drug discovery for many human diseases, the interdependence of each signaling pathway may be an obstacle to the development of a nonpeptide orally active small molecule to inhibit one of these IL-6 signaling cascades, because it would disturb the signal orchestration. In mice, a consequence of the imbalanced signals causes unexpected results such as gastrointestinal disorders, autoimmune diseases, and/or chronic inflammatory proliferative diseases. However, lessons learned from IL-6 KO mice indicate that IL-6 is not essential for vital biological processes, but a significant impact on disease progression in many experimental models for human disorders. Thus, IL-6/gp130 signaling will become a more attractive therapeutic target for human inflammatory diseases when a better understanding of IL-6 signaling, including the identification of the conductor for gp130 signal transduction, is achieved.

Effects of fasting on the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis were examined in the tilapia ( Oreochromis mossambicus ) acclimated to fresh water. Fasting for 2 weeks resulted in significant reductions in body weight, specific growth rate and hepatosomatic index in both males and females. Significant reductions in specific growth rates were observed after 1 and 2 weeks in both sexes, although the decrease in body weight was not significant in the female. A significant reduction was also seen in the condition factor of females after 2 weeks. No change was seen in the gonadosomatic index in either sex. Two weeks of fasting also produced a significant reduction in plasma IGF-I but not in plasma GH, prolactin (PRL 188 ) or cortisol. Significant reductions in the hepatic IGF-I mRNA were seen in both sexes. On the other hand, a significant increase was observed in cortisol receptor mRNA in the female liver. Plasma IGF-I levels were correlated significantly with specific growth rate, condition factor and hepatosomatic index, indicating that plasma IGF-I is a good indicator of growth in the tilapia. No change was seen in plasma glucose or osmolality after 2 weeks of fasting. During fasting, tilapia appears to convert metabolic energy from growth to basal metabolism including maintenance of ion and water balance.

We examined the effects of one-year high-dose bisphosphonates (risedronate 0.5 mg/kg/day or alendronate 1.0 mg/kg/day) on the three-dimensional (3-D) microstructural and mechanical properties of canine cancellous bone. A high-resolution micro-CT scanner was used to scan cubic specimens produced from the first lumbar vertebrae. Microstructural properties of the specimens were calculated directly from the 3-D datasets and the mechanical properties of the specimens were determined. Our data demonstrate significant microarchitectural changes in the bisphosphonate-treated cancellous bone that was typically plate-like, denser, with thicker and more trabeculae compared with those of the controls. Consistent with architectural changes, the Young's moduli of cancellous bone increased in all three directions with the greatest increase in primary axial loading (cephalo-caudal) direction after treatment. Our results suggest a bone remodeling-adaptation mechanism stimulated by bisphosphonates that increases bone volume fraction, thickens trabeculae, changes trabeculae towards more plate-like, and increases mechanical properties. The secondary degree of anisotropy contributed significantly to the explained variance in bone strength, and the primary or tertiary degree of anisotropy improved the explanation of variances for Young's moduli, i.e., 79% of strength variances or 74-83% of modulus variances could be explained by the combined anisotropy and bone volume fraction. These significant improvements of cancellous bone architecture provide a rationale for the clinical observation that fracture risk decreased by 50% in the first year of bisphosphonate therapy with only a 5% increase in bone mineral density. We conclude that bisphosphonates enhance mechanical properties and reduce fracture risk by improving architectural anisotropy of cancellous bone 3-D microarchitecture.

Bovine claudin-16/paracellin-1 (CL-16/PCLN-1) deficiency is an autosomal recessive disorder caused by a 37-kb deletion mutation containing the first four exons of the CL-16 gene, which leads to the absence of the CL-16 transcript (type-1 mutation). A PCR-based DNA test for the CL-16 mutation (type-1) was used to screen a herd of Wagyu cattle. A recent report suggested that affected cattle can be bred by dams diagnosed as normal, suggesting the presence of a new mutation in the CL-16 locus. We identified the new mutation as a 56-kb deletion containing exon-1 to -4 and 21-bp of exon-5 of CL-16, and refer to this as a type-2 mutation. A DNA test for specific for this mutation was then established.

Two PRL-releasing peptides (PrRP20 and PrRP31) were recently identified from mammalian hypothalamus by an orphan receptor strategy, and a C-terminal RF (arginyl-phenylalamyl-) amide peptide (RFa), structurally related to mammalian PrRP, was also identified from the brain of the Japanese crucian carp (C-RFa) by an intestine-contracting assay. However, to date there have been no reported studies that have examined the PRL-releasing effects of RFa in fish. In the present study we determined the cDNA, primary structure, and function of a homolog of the mammalian PrRP20 in the chum salmon, Oncorhynchus keta. An RFa cDNA encoding a preprohormone of 155 amino acids was cloned from the hypothalamus of chum salmon by 3'- and 5'-rapid amplification of cDNA ends. A native RFa was purified from an acid extract of salmon hypothalami by a Sep-Pak C(18) cartridge, affinity chromatography using anti-synthetic C-RFa, and reverse phase HPLC on an ODS-120T column. The salmon RFa proved to be identical with C-RFa on the basis of elution position on reverse phase HPLC. Immunocytochemical staining in rainbow trout, Oncorhynchus mykiss, revealed that C-RFa-immunoreactive cell bodies were located in the posterior part of hypothalamus and C-RFa-immunoreactive fibers were abundant from the hypothalamus to the ventral telencephalon. A small number of immunoreactive fibers were projected to the pituitary and terminated close to the PRL cells in the rostral pars distalis and to the somatolactin (SL) cells in the pars intermedia. The hypophysiotropic effects of the fish homolog were determined on the release of PRL, SL, and GH from the pituitary of the rainbow trout. Plasma PRL and SL levels were increased at 3 and 9 h, respectively, after ip injection of the synthetic C-RFa into the rainbow trout at doses of 50 and 500 ng/g body weight. In contrast, plasma GH levels were decreased after 1 h at 500 ng/g body weight. Perifusion of the trout pituitaries with synthetic C-RFa at concentrations of 10 pM to 100 nM demonstrated maximum PRL release at 100 pM and maximum SL release at 10 and 100 nM. However, GH release was not affected. These data are the first to demonstrate that a homolog of mammalian PrRP (fish RFa) is a major hypothalamic peptide of PRL release in teleost fish.

This study was designed to evaluate the effect of anticancer agent 5-fluorouracil (5-FU)) on the exocrine pancreas by using isolated perfused rat pancreas at three different doses (2.5, 5.0 and 10 microg/ml). 5-FU at doses of 5 and 10 microg/ml for 1 h caused significant increases in amylase levels in pancreatic effluent and significant decreases in amylase outputs. Moreover, 5-FU caused significant increases in pancreatic trypsin contents in pancreatic tissue. 5-FU also caused significant redistribution of cathepsin B activity from the lysosomal fraction to the zymogen fraction in pancreatic subcellular fractionation study. These changes were almost dose-dependent. These results suggest that the exocrine pancreas might be injured by 5-FU, and that lysosomal enzymes seem to play an important role in the pathogenesis of the pancreatic injuries induced by 5-FU, since cathepsin B can activate trypsinogen.

We have previously reported an infiltration of renal interstitial gammadelta T cells in Adriamycin-induced progressive glomerulosclerosis in the rat kidney. The TCR repertoire and sequences used by these gammadelta T cells have now been studied. Two injections of Adriamycin 14 days apart caused segmental glomerulosclerosis, massive interstitial infiltration of mononuclear cells, and end-stage renal failure. Flow cytometry of lymphocyte subpopulations with Abs to CD3, the gammadelta TCR, and the alphabeta TCR showed that gammadelta T cells as a proportion of CD3(+) cells were increased in Adriamycin-treated kidneys (8.5 +/- 5.4%), but not in lymph nodes (1.3 +/- 0.4%). A semiquantitative score of glomerular damage (r = 0.65; p0.01) and creatinine (r = 0.62; p0.01) correlated significantly with the presence of gammadelta T cells. TCR Vgamma repertoire analysis by RT-PCR and Southern blotting showed that Vgamma2 was the dominant subfamily in lymph nodes, whereas Vgamma4 became the predominant subfamily in advanced stages of the rat Adriamycin-treated kidney. Sequencing of the Vgamma4-Jgamma junctional region showed an invariant sequence. The amino acid sequence of the junctional region of the Vgamma4 TCR was the same as the reported mouse canonical Vgamma4 TCR sequence. Analysis of the kidney Vdelta repertoire showed dominant expression of Vdelta1, and sequencing again revealed the selective expression of a canonical Vdelta1 gene. Semiquantitative RT-PCR for cytokine gene expression showed that gammadelta T cells from the kidneys expressed TGF-beta, but not IL-4, IL-10, or IFN-gamma. These results suggest that the predominant gammadelta T cells in the Adriamycin kidney use an invariant Vgamma4/Vdelta1 receptor.

We recently demonstrated that suppression of bone remodeling allows microdamage to accumulate, leading to reduced bone toughness in the rib cortex of dogs. This study evaluates the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties at clinically relevant skeletal sites in the same dogs. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle (CNT). The remaining two groups were treated daily with risedronate at a dose of 0.5 mg/kg per day (RIS), or alendronate at 1.0 mg/kg per day (ALN) orally. The doses of these bisphosphonates were six times the clinical doses approved for treatment of osteoporosis in humans. After killing, the L-1 vertebra was scanned by dual-energy X-ray absorptiometry (DXA), and the L-2 vertebra and right ilium were assigned to histomorphometry. The L-3 vertebra, left ilium, Th-2 spinous process, and right femoral neck were used for microdamage analysis. The L-4 vertebra and Th-1 spinous process were mechanically tested to failure in compression and shear, respectively. One year treatment with risedronate or alendronate significantly suppressed trabecular remodeling in vertebrae (RIS 90%, ALN 95%) and ilium (RIS 76%, ALN 90%) without impairment of mineralization, and significantly increased microdamage accumulation in all skeletal sites measured. Trabecular bone volume and vertebral strength increased significantly following 12 month treatment. However, normalized toughness of the L-4 vertebra was reduced by 21% in both RIS (p = 0.06) and ALN (p = 0.05) groups. When the two bisphosphonate groups were pooled in a post hoc fashion for analysis, this reduction in toughness reached statistical significance (p = 0.02). This study demonstrates that suppression of trabecular bone turnover by high doses of bisphosphonates is associated with increased vertebral strength, even though there is significant microdamage accumulation and a reduction in the intrinsic energy absorption capacity of trabecular bone.

This study was designed to evaluate the protective effect of thromboxane A2 (TXA2) receptor antagonist, seratrodast, against pancreatic injuries during acute pancreatitis. Acute pancreatitis was induced in rats by intravenous infusion of a supramaximal dose of caerulein (5 microg/kg x h for 4 h). In this model, marked hyperamylasemia, a significant increase in pancreatic water content, and a significant increase in pancreatic micro-vascular leakage of Evans blue dye were observed. Pancreatic subcellular redistribution of a lysosomal enzyme, cathepsin B from the lysosomal fraction to the zymogen fraction as well as a significant increase in pancreatic trypsin content were also observed. Pretreatment with seratrodast at a dose of 2 mg/kg (twice, 8 and 4 h before caerulein infusion) significantly inhibited these pancreatic injuries including hyperamylasemia, increased pancreatic microvascular leakage, redistribution of cathepsin B and increased pancreatic trypsin content. These results suggest that TXA2 may be involved in the pathogenesis of acute pancreatitis in the early stage of the disease and that TXA2 receptor antagonist might be of therapeutic value for treatment of acute pancreatitis.

The establishment of sister chromatid cohesion during S phase and its dissolution at the metaphase-anaphase transition are essential for the faithful segregation of chromosomes in mitosis [1-4]. Recent studies in yeast genetics and Xenopus biochemistry have identified a large protein complex, cohesin, that plays a key role in sister chromatid cohesion [5-10]. The cohesin complex consists of a heterodimeric pair of SMC (structural maintenance of chromosomes) subunits and at least two non-SMC subunits. This structural organization is reminiscent of that of condensin, another major SMC protein complex that drives chromosome condensation in eukaryotic cells [11]. Condensin has been shown to reconfigure and compact DNA in vitro by utilizing the energy of ATP hydrolysis [12]. Very little is known, however, about how cohesin works at a mechanistic level. Here we report the first set of biochemical activities associated with an intact cohesin complex purified from HeLa cell extracts. The cohesin complex binds directly to double-stranded DNA and induces the formation of large protein-DNA aggregates. In the presence of topoisomerase II, cohesin stimulates intermolecular catenation of circular DNA molecules. This activity is in striking contrast to intramolecular knotting directed by condensin [13]. Cohesin also increases the probability of intermolecular ligation of linear DNA molecules in the presence of DNA ligase. Our results are consistent with a model in which cohesin functions as an intermolecular DNA crosslinker and is part of the molecular "glue" that holds sister chromatids together [14].

We developed a novel method for the chemical modification of cytokines with synthetic polymers to increase the therapeutic efficacy of the former in vivo. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), was used for modification of interleukin-6 (IL-6) with polyethylene glycol (PEG). The novel PEG-conjugated IL-6 (DmPEG-IL-6), which had been pretreated with DMMAn before PEGylation, showed up to a 140% increase in in vitro specific activity compared with PEG-IL-6 that had been synthesized by the previous method. Moreover, DmPEG-IL-6 caused thrombopoiesis more potently in mice than PEG-IL-6. The DmPEG-IL-6 Fr.1, having 3-4 PEG chains attached to the cytokine, showed the strongest thrombopoietic effect among the DmPEG-IL-6s with different molecular sizes that were tested. PEG-IL-6 Fr.1 had a 500-fold higher potency in stimulating thrombopoiesis than native IL-6 and DmPEG-IL-6 Fr.1 achieved a threefold higher thrombopoietic effect than PEG-IL-6 Fr.1. In addition, side-effects, such as an increase in the plasma fibrinogen level, were not observed after injection of either PEG-IL-6s or DmPEG-IL-6s. These results suggest that PEGylation with DMMAn pretreatment may become a useful means for clinical cytokine delivery.

Cortical porosity in patients with hyperparathyroidism has raised the concern that intermittent parathyroid hormone (PTH) given to treat osteoporotic patients may weaken cortical bone by increasing its porosity. We hypothesized that treatment of ovariectomized (OVX) cynomolgus monkeys for up to 18 months with recombinant human PTH(1-34) [hPTH(1-34)] LY333334 would significantly increase porosity in the midshaft of the humerus but would not have a significant effect on the strength or stiffness of the humerus. We also hypothesized that withdrawal of PTH for 6 months after a 12-month treatment period would return porosity to control OVX values. OVX female cynomolgus monkeys were given once daily subcutaneous (sc) injections of recombinant hPTH(1-34) LY333334 at 1.0 microg/kg (PTH1), 5.0 microg/kg (PTH5), or 0.1 ml/kg per day of phosphate-buffered saline (OVX). Sham OVX animals (sham) were also given vehicle. After 12 months, PTH treatment was withdrawn from half of the monkeys in each treatment group (PTH1-W and PTH5-W), and they were treated for the remaining 6 months with vehicle. Double calcein labels were given before death at 18 months. After death, static and dynamic histomorphometric measurements were made intracortically and on periosteal and endocortical surfaces of sections from the middiaphysis of the left humerus. Bone mechanical properties were measured in the right humeral middiaphysis. PTH dose dependently increased intracortical porosity. However, the increased porosity did not have a significant detrimental effect on the mechanical properties of the bone. Most porosity was concentrated near the endocortical surface where its mechanical effect is small. In PTH5 monkeys, cortical area (Ct.Ar) and cortical thickness (Ct.Th) increased because of a significantly increased endocortical mineralizing surface. After withdrawal of treatment, porosity in PTH1-W animals declined to sham values, but porosity in PTH5-W animals remained significantly elevated compared with OVX and sham. We conclude that intermittently administered PTH(1-34) increases intracortical porosity in a dose-dependent manner but does not reduce the strength or stiffness of cortical bone.

OPC-51803 is the first nonpeptide vasopressin (AVP) V(2)-receptor-selective agonist. Its pharmacological profile, including antidiuretic action and receptor binding, was characterized using conscious Brattleboro rats with hereditary diabetes insipidus and Sprague-Dawley rats. In membrane preparations from the liver and kidney, OPC-51803 displaced the [(3)H]AVP binding to V(2)-receptors (K(i) = 49.8 +/- 8.1 nM) more greatly than that to V(1a)-receptors (K(i) = 1061 +/- 60 nM), showing a 21 times higher affinity for V(2)-receptors. At single oral doses of 0.003 to 0.3 mg/kg in female Brattleboro rats, OPC-51803 decreased urine volume (from 10.8 +/- 1.1 to 0.5 +/- 0.2 ml during 0-2 h postdosing) and increased urinary osmolality (from 114 +/- 9 to 432 +/- 114 mOsm/kg) in a dose-dependent manner. During the period of 4-week treatment with OPC-51803, significant and constant antidiuresis was observed. In male Sprague-Dawley rats with normal plasma AVP levels, OPC-51803 at 0.03 to 0.3 mg/kg also produced a dose-dependent antidiuretic action (urine volume: from 2.6 +/- 0.6 to 1.1 +/- 0.2 ml at 0-4 h postdosing). Few changes in urinary parameters, serum parameters, or plasma hormone levels were observed. OPC-51803 did not change blood pressure or heart rate, or inhibit AVP-induced pressor response even at 30 mg/kg p.o. These results demonstrate that OPC-51803 is a V(2)-selective agonist that produces a significant antidiuretic action after single and multiple oral dosing in AVP-deficient and normal AVP states. The data suggest that OPC-51803 is a useful therapeutic drug in the treatment of hypothalamic diabetes insipidus, nocturnal enuresis, and some kinds of urinary incontinence.

Cleavage is one of the initial steps of embryogenesis, and is characterized by a series of symmetric and synchronous cell divisions. We showed that p38 MAP kinase (p38) is asymmetrically activated on one side of the blastodisc during the early cleavage period in zebrafish (Danio rerio) embryos. When a dominant negative (DN) form of p38 was uniformly expressed, blastomere cleavage was impaired on one side of the blastodisc, resulting in the formation of blastomeres with a large mass of cytoplasm and an enlarged nucleus on the affected side. The area affected by the DN-p38 expression did not correlate with the initial cleavage plane, but coincided with the side where dharma/bozozok, a dorsal-specific zygotic gene, was expressed (Yamanaka et al. 1998). Furthermore, UV irradiation and removal of the vegetal yolk mass before the first cleavage, both of which inhibit the initiation of the dorsalizing signals, abolished the asymmetric p38 activation. Our findings suggest that asymmetric p38 activation is required for symmetric and synchronous cleavage, and may be regulated by the same machinery that controls the initiation of dorsalizing signals.