Hepatogenous photosensitization associated with liver copper accumulation in buffalos

Four 1.5-year-old, male, Murrah buffalos were maintained during eight months without direct solar exposure during a study of copper toxicosis. Four days after solar exposure, all buffalos presented clinical manifestations consistent with acute photosensitization, including anorexia, apathy, loss of body weight, and generalized cutaneous lesions. Gross lesions were characterized by severe erythema, localized edema, fissures, tissue necrosis, gangrene and crust formation with h serous exudation. Liver copper concentration was evaluated, and cutaneous biopsies were taken when clinical signs were evident. The liver copper concentration before solar exposure was increased in all animals. Histopathologic examination of the skin revealed hepatogenous photosensitization characterized by parakeratotic hyperkeratosis, acantholysis, degeneration of squamous epithelial cells, epidermal necrosis with atrophy of sweat glands, and multifocal superficial and deep dermal edema. These findings suggest that asymptomatic accumulation of copper within the liver might have induced hepatic insufficiency thereby resulting in secondary photosensitization when these buffalos were exposed to sunlight. (C) 2009 Elsevier Ltd. All rights reserved.

Alteration of Ca(2+)-ATPase activity in the homogenate, plasma membrane and microsomes of the salivary glands of streptozotocin-induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca(2+)-ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state oil this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH(4)Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca(2+)-ATPase (total, independent, and dependent) was determined in the homo-enate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be hi-her in the diabetic animals. Ca(2+)-ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright (c) 2009 John Wiley & Sons, Ltd.

Healing of critical-size cranial defects in guinea pigs using a bovine bone-derived resorbable membrane

Purpose: To investigate the healing of critical-size cranial bone defects (9-mm-diameter) in guinea pigs treated with a bovine bone-derived resorbable membrane. Materials and Methods: A sample of 42 guinea pigs was divided into test (n = 20), control (n = 20), and standard (n = 2) groups. A full-thickness trephine defect was made in the fronto-parietal bone of each animal. In the test group, the internal and external openings of the defect were each closed with a separate membrane, and the space between them was filled with blood clot and a central spacer. In the control group, the defect was filled only with the blood clot and spacer. At 1, 3, 6, and 9 months later, the calvarias (5 per period) for both the test and control groups were collected, fixed, radiographed, and histologically processed. The Standard-group animals were sacrificed immediately after surgery and used to determine the initial size of defect radiographically. The areas of defects in the radiographs were measured with image-analysis software and were compared between groups and periods by multiple regression analysis with the Bonferroni correction. Results: At 1 and 3 months, newly formed woven bone was histologically observed in both test and control groups. Radiographically, this new bone occupied an average of 32% of the defect area at 1 month and 60% at 3 months in the test group. In the control group, 21% of the defect was filled at 1 month and 39% at 3 months. However, the differences between treatments were not statistically significant (P > .05). At 6 and 9 months, a significant increase in newly formed lamellar bone was seen histologically in both groups. Radiographically, for the test group, the new bone occupied an average of 82% of the defect area at 6 months and 96% at 9 months. For the control group, new bone composed an average of 45% of the defect area at 6 months and 40% at 9 months. The differences between the test and control groups were statistically significant at 6 and 9 months (P < .05). Complete or almost complete filling of the defect was observed in several cases. Conclusion: It was concluded that the bovine bone-derived membrane is highly biocompatible and is able to promote good healing of critical-size defects in calvaria of guinea pig.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.

A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: Evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of V beta T cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR V beta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

Recent advances in the pathology of alcoholic myopathy

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Victor R. Preedy and Junko Adachi. The presentations were (1) Alcoholic myopathy: Past, present and future, by Timothy J. Peters and Victor R. Preedy; (2) Protein adducts in the type I and II fiber-predominant muscles of the ethanol-fed rat, by Simon Worrall, Seppo Parkkila, and Onni Niemela; (3) Hydroperoxides and changes in alcoholic myopathy, by Junko Adachi, Migiwa Asamo, and Yasuhino Ueno; and (4) A close association between testicular atrophy, muscle atrophy, and the increase in protein catabolism after chronic ethanol administration, by Kunihiko Takeda, Masayoshi Yamauchi, Kazuhiko Sakamoto, Masaru Takagi, Hisato Nakajima, and Gotaro Toda.

Disposition of naproxen, naproxen acyl glucuronide and its rearrangement isomers in the isolated perfused rat liver

1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 mug NAP equivalents ml(-1) perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t(1/2)) of 13.4 +/-4.4 h. No metabolites were detected in perfusate, while NAG was the only metabolite present in bile in measurable amounts (3.9 +/-0.8%, of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t(1/2) in perfusate=57 +/-3 and 75 +/- 14min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results Suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 kg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 mug/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYPIA2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYPIA2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, WIC, and CIC were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations. (C) 2001 Elsevier Science Inc. All rights reserved.

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): A comparison with their in situ counterparts

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in p-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha -actinin are organized into longitudinally arranged myofibrils and the vimentin-containing intermediate filaments form a meshed cytoskeletal network, However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins. (C) 2001 Academic Press.

Population pharmacokinetics of tacrolimus in children who receive cut-down or full liver transplants

Background. The aim of this study was to investigate the population pharmacokinetics of tacrolimus in pediatric liver transplant recipients and to identify factors that may explain pharmacokinetic variability. Methods. Data were collected retrospectively from 35 children who received oral immunosuppressant therapy with tacrolimus. Maximum likelihood estimates were sought for the typical values of apparent clearance (CL/F) and apparent volume of distribution (V/F) with the program NONMEM. Factors screened for influence on the pharmacokinetic parameters were weight, age, gender, postoperative day, days since commencing tacrolimus therapy, transplant type (whole child liver or cut-down adult liver), liver function tests (bilirubin, alkaline phosphatase [ALP], aspartate aminotransferase [AST], gamma -glutamyl transferase [GGT], alanine aminotransferase [ALT]), creatinine clearance, hematocrit, corticosteroid dose, and concurrent therapy with metabolic inducers and inhibitors of tacrolimus. Results. No clear correlation existed between tacrolimus dosage and blood concentrations (r(2) =0.003). Transplant type, age, and liver function test values were the most important factors (P

Prevention of intraabdominal adhesions in Kasai portoenterostomy

An infant with biliary atresia had the right side of his liver covered with a sodium hyaluronate-based bioresorbable membrane during his initial Kasai portoenterostomy procedure. When his peritoneal cavity was entered 10.6 months (317 days) later for a liver transplant operation there was a remarkable absence of intraabdominal adhesions leading to a smooth operation and an uncomplicated recovery. J Pediatr Surg 36:1613-1614. Copyright (C) 2001 by W.B. Saunders Company.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.