Changes of antioxidant enzymes in the kidney after cardiac arrest in the rat model.

Globally, cardiac arrest (CA) is a leading cause of death and disability. Asphyxial CA (ACA)-induced kidney damage is a crucial factor in reducing the survival rate. The purpose of this study was to investigate the role of antioxidant enzymes in histopathological renal damage in an ACA rat model at different time points. A total of 88 rats were divided into five groups and exposed to ACA except for the sham group. To evaluate glomerular function and oxidative stress, serum levels of blood urea nitrogen (BUN) and creatinine (Crtn) and malondialdehyde (MDA) levels in renal tissues were measured. To determine histopathological damage, hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson's trichrome staining were performed. Expression levels of antioxidant enzymes including superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), catalase (CAT), and glutathione peroxidase (GPx) were measured by immunohistochemistry (IHC). Survival rate of the experimental rats was reduced to 80% at 6 h, 55% at 12 h, 42.9% at 1 day, and 33% at 2 days after return of spontaneous circulation. Levels of BUN, Crtn, and MDA started to increase significantly in the early period of CA induction. Renal histopathological damage increased markedly from 6 h until two days post-CA. Additionally, expression levels of antioxidant enzymes were significantly decreased at 6 h, 12 h, 1 day, and 2 days after CA. CA-induced oxidative stress and decreased levels of antioxidant enzymes (SOD-1, SOD-2, CAT, GPx) from 6 h to two days could be possible mediators of severe renal tissue damage and increased mortality rate.

Changes of antioxidant enzymes in the kidney after cardiac arrest in the rat model

Globally, cardiac arrest (CA) is a leading cause of death and disability. Asphyxial CA (ACA)-induced kidney damage is a crucial factor in reducing the survival rate. The purpose of this study was to investigate the role of antioxidant enzymes in histopathological renal damage in an ACA rat model at different time points. A total of 88 rats were divided into five groups and exposed to ACA except for the sham group. To evaluate glomerular function and oxidative stress, serum levels of blood urea nitrogen (BUN) and creatinine (Crtn) and malondialdehyde (MDA) levels in renal tissues were measured. To determine histopathological damage, hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson's trichrome staining were performed. Expression levels of antioxidant enzymes including superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), catalase (CAT), and glutathione peroxidase (GPx) were measured by immunohistochemistry (IHC). Survival rate of the experimental rats was reduced to 80% at 6 h, 55% at 12 h, 42.9% at 1 day, and 33% at 2 days after return of spontaneous circulation. Levels of BUN, Crtn, and MDA started to increase significantly in the early period of CA induction. Renal histopathological damage increased markedly from 6 h until two days post-CA. Additionally, expression levels of antioxidant enzymes were significantly decreased at 6 h, 12 h, 1 day, and 2 days after CA. CA-induced oxidative stress and decreased levels of antioxidant enzymes (SOD-1, SOD-2, CAT, GPx) from 6 h to two days could be possible mediators of severe renal tissue damage and increased mortality rate.

Branching patterns of the subclavian arteries in German Shepherd dogs.

This study is the first report of the branching pattern of the four major branches of the subclavian artery in German Shepherd dogs. A total 116 subclavian artery casts made of silicon under mean arterial pressure were analysed. The casts were classified according to their branching order and the pattern of the first two branches of the vertebral artery and costocervical trunk. The three subtypes of each major type were based on the branching order or level of the next two branches (the superficial cervical artery and internal thoracic artery). Eleven of 12 possible subtypes were found in the samples. The number of dogs having the same branching subtype of the left and right subclavian arteries was not greater than those having a different subtype. The distance between the first and last branches of the subclavian artery was always longer on the left side than on the right. However, gender-based differences were identified neither in the subtype patterns nor in the distance between the branches. These results suggest a variable branching of the subclavian arteries with frequent differences of the branching pattern on the left and right sides in German Shepherds.

The haploinsufficient Col3a1 mouse as a model for vascular Ehlers-Danlos syndrome.

Vascular Ehlers-Danlos syndrome is a rare genetic disorder resulting from mutations in the α-1 chain of type III collagen (COL3A1) and manifesting as tissue fragility with spontaneous rupture of the bowel, gravid uterus, or large or medium arteries. The heterozygous Col3a1 knockout mouse was investigated as a model for this disease. The collagen content in the abdominal aorta of heterozygotes was reduced, and functional testing revealed diminishing wall strength of the aorta in these mice. Colons were grossly and histologically normal, but reduced strength and increased compliance of the wall were found in heterozygotes via pressure testing. Although mice demonstrated no life-threatening clinical signs or gross lesions of vascular subtype Ehlers-Danlos syndrome type IV, thorough histological examination of the aorta of heterozygous mice revealed the presence of a spectrum of lesions similar to those observed in human patients. Lesions increased in number and severity with age (0/5 [0%] in 2-month-old males vs 9/9 [100%] in 14-month-old males, P < .05) and were more common in male than female mice (23/26 [88.5%] vs 14/30 [46.7%] in 9- to 21-month-old animals, P < .05). Haploinsufficiency for Col3a1 in mice recapitulates features of vascular Ehlers-Danlos syndrome in humans and can be used as an experimental model.

Morphometric studies on the testis of Korean ring-necked pheasant (Phasianus colchicus karpowi) during the breeding and non-breeding seasons.

The purpose of this study was to obtain detailed quantitative information on all cell types in the testis interstitium of Korean ring-necked pheasants and to combine these data with changes in the steroidogenic function of the testis during the breeding and non-breeding seasons. For animals collected during the breeding season, their testis weights, sperm production, serum testosterone levels and leuteinizing hormone (LH)-stimulated testosterone secretion were significantly (p < 0.01) increased compared to the non-breeding season. Testes of the pheasants during the non-breeding season displayed a 98% reduction in testis volume that was associated with a decrease in the absolute volume of seminiferous tubules (98% reduction), tubular lumen (100%), interstitium (90%), blood vessels (84%), lymphatic spaces (97%), Leydig cells (79%), mesenchymal cells (51%) and myoid cells (61%) compared to the breeding season. The numbers of Leydig cells, mesenchymal cells and myoid cells per testis in the breeding season were much higher than in the non-breeding season. Although the mean volume of a Leydig cell was 74% lower in the non-breeding season, the mean volumes of myoid and mesenchymal cells remained unchanged. These results demonstrate that there are striking differences in the testicular structure of the Korean ring-necked pheasant during the breeding and non-breeding seasons. Every structural parameter of the Leydig cell was positively correlated with both testosterone serum levels and LH-stimulated testosterone secretion. The correlation of changes in hormonal status with the morphometric alterations of Leydig cells suggests that the Korean-ring necked pheasant may be used as a model to study structure-function relationships in the avian testis.

Impact of circulating bone-resorbing cytokines on the subsequent bone loss following bone marrow transplantation.

Cytokines including IL-6 and TNF-alpha play an important role in the pathogenesis of postmenopausal osteoporosis. However, the relationship between changes in the cytokine levels and subsequent bone loss in patients undergoing a bone marrow transplantation (BMT) is unclear. A total of 46 patients undergoing an allogeneic BMT were prospectively investigated. The bone turnover markers and the serum cytokines were measured before BMT and serially after BMT. Bone mineral density (BMD) was measured before and 1 year after BMT. At 1 year after BMT, the lumbar spine BMD had decreased by 4.8%, and the total proximal femoral BMD had decreased by 12.3%. The serum IL-6 and TNF-alpha levels increased until 2 and 3 weeks after BMT, respectively. The lumbar BMD was significantly decreased as the serum IL-6 and TNF-alpha levels increased by post-BMT 3 weeks. The lumbar BMD decreased significantly as the cumulative prednisolone and cyclosporine dose increased. Patients with GVHD > or =grade II had higher lumbar bone loss than patients with GVHD

B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos.

B-ATF is a nuclear basic leucine zipper protein that belongs to the AP-1/ATF superfamily of transcription factors. Northern blot analysis reveals that the human B-ATF gene is expressed most highly in hematopoietic tissues. Interaction studies in vitro and in vivo show that the leucine zipper of B-ATF mediates dimerization with members of the Jun family of proteins. Chimeric proteins consisting of portions of B-ATF and the DNA binding domain of the yeast activator GAL4 do not stimulate reporter gene expression in mammalian cells, indicating that B-ATF does not contain a conventional transcription activation domain. Jun/B-ATF dimers display similar DNA binding profiles as Jun/Fos dimers, with a bias toward binding TRE (12-O-tetradecanolyphorbol-13-acetate-response element) over CRE (cyclic AMP-response element) DNA sites. B-ATF inhibits transcriptional activation of a reporter gene containing TRE sites in a dose-dependent manner, presumably by competing with Fos for Jun and forming transcriptionally inert Jun/B-ATF heterodimers. Stable expression of B-ATF in C3H10T1/2 cells does not reduce cell viability, but does result in a reduced cellular growth rate when compared to controls. This effect is dominant in the presence of the growth promoting effects of the H-Ras or the v-Fos oncoproteins, since expression of B-ATF restricts the efficiency of focus formation by these transforming agents. These findings demonstrate that B-ATF is a tissue-specific transcription factor with the potential to function as a dominant-negative to AP-1.

IFP 35 forms complexes with B-ATF, a member of the AP1 family of transcription factors.

Multicellular organisms achieve the spatial and temporal regulation of genes during growth and development through the differential expression of transcription factors that associate in various combinations. In this paper, we report the physical association of B-ATF, a member of the AP1 family of basic leucine zipper transcription factors, with IFP 35, a leucine zipper protein that is translocated to the nucleus following the treatment of cells with interferons and for which no binding partners previously have been described. Our data suggest that the formation of B-ATF:IFP 35 heterodimers is an interferon-inducible event in specialized cell types expressing both proteins and that changes in AP1 mediated gene transcription likely play a role in the response of these cells to interferons.

Regulation of acetyl coenzyme-A carboxylase gene in a transgenic animal model.

The gene for acetyl-CoA carboxylase, the rate-limiting enzyme in the biosynthesis of long-chain fatty acids, contains two promoters; promoter I (PI) and promoter II (PII) which are responsible for generation of class I and class II ACC mRNAs, respectively. Class I ACC mRNAs are present in adipose tissue, but only a trace was found in the liver under normal physiological conditions. However, class I mRNAs were induced under stimulated lipogenic conditions. To investigate how PI is regulated in vivo, we generated transgenic mice containing a reporter gene under the control of PI. In transgenic mice, PI is generally inactive and a small amount of PI activity was found only in the adipose tissues of female animals. Stimulated lipogenic conditions activated PI about 17-fold over normal conditions and again only in white adipose tissues of female animals.

B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family.

A new member of the ATF/CREB family of transcription factors, called B-ATF, has been isolated from a cDNA library prepared from Epstein-Barr virus stimulated human B cells. B-ATF is a 125 amino acid nuclear protein possessing a basic leucine zipper domain that is most similar to the basic leucine zipper of ATF-3. Northern blot analysis of polyadenylated mRNA isolated from a variety of human tissues and established cell lines indicates that the 1.0 kilobase B-ATF mRNA is expressed differentially, with the strongest hybridization detected in lung and in Raji Burkitt's lymphoma. Efficient homodimerization of the B-ATF protein cannot be detected using the yeast two hybrid system or using in vitro binding assays with glutathione-s-transferase-B-ATF and maltose binding protein-B-ATF fusion proteins produced in E. coli. However, a yeast two hybrid library screen has identified the human oncoprotein JunB as a specific binding partner for B-ATF. Glutathione-s-transferase-B-ATF heterodimerizes efficiently with in vitro translated JunB, c-Jun, and JunD, but only weakly associates with c-Fos. In addition, electrophoretic mobility shift assays demonstrate that a B-ATF/c-Jun protein complex can interact with DNA containing a consensus binding site for AP-1, suggesting that B-ATF functions as a tissue-specific modulator of the AP-1 transcription complex in human cells.

cAMP activation of CAAT enhancer-binding protein-beta gene expression and promoter I of acetyl-CoA carboxylase.

The acetyl-CoA carboxylase (ACC) gene contains two distinct promoters, denoted PI and PII. PI is responsible for the generation of class I ACC mRNAs which are induced in a tissue-specific manner under lipogenic conditions. PII generates class II ACC mRNAs which are expressed constitutively. During 30A5 preadipocyte differentiation, both promoters are activated; the preadipocytes must be pretreated with cAMP for this activation to occur. In this report, we present evidence that CAAT enhancer-binding protein-beta (C/EBP-beta) is induced and involved in the PI activation by cAMP. Expression of the reporter gene under the control of the PI promoter is activated within 3 h after treatment of 30A5 cells with a cyclic AMP analogue, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, and 3-isobutyl-1-methylxanthine, in association with the accumulation of C/EBP-beta mRNA and protein. These accumulations were inhibited in the presence of H8, a protein kinase inhibitor; H8 also inhibited activation of PI by cAMP. However, the induction of reporter gene expression and the increase of C/EBP-beta mRNA by cAMP were not affected by treatment with tumor necrosis factor alpha, which completely inhibited the accumulation of C/EBP-alpha mRNA. Overexpression of C/EBP-beta by transfection with the C/EBP-beta gene led to increased binding of C/EBP-beta to DNA and partial PI activation. cAMP did not affect the amount of C/EBP-beta binding to the DNA but did promote phosphorylation of C/EBP-beta and PI activation. As in the case of C/EBP-alpha, C/EBP-beta bound to the CCAAT box of the PI promoter. These results indicate that cAMP not only induces, but also activates, bound C/EBP-beta through phosphorylation for PI activation. Our studies also indicate that cAMP induces C/EBP-alpha. C/EBP-beta induction, however, precedes that of C/EBP-alpha.

Pattern and regulation of acetyl-CoA carboxylase gene expression.

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long chain fatty acids. There is a single copy of the gene for acetyl-CoA carboxylase per haploid chromosome set. The gene contains two promoters whose primary transcripts are differentially spliced resulting in multiple forms of acetyl-CoA carboxylase mRNA. These mRNA species are different in the 5'-untranslated region, but contain the same coding region. Generation of different forms of the mRNA is tissue specific and controlled by physiological conditions. Two promoters contain an extensive array of cis-elements that perceive changes in the cellular environment signalling repression and induction of long chain fatty acid synthesis. The ability of the gene to respond to various lipogenic signals and the presence of the same coding sequence in all acetyl-CoA carboxylase mRNA species suggest that the biosynthesis of fatty acids required for multiple functions in the cells is primarily regulated at the gene level.

Roles of CCAAT/enhancer-binding protein and its binding site on repression and derepression of acetyl-CoA carboxylase gene.

The gene for acetyl-CoA carboxylase, the rate-limiting enzyme in the biosynthesis of long-chain fatty acids, contains two distinct promoter regions, denoted PI and PII, which control the generation of different forms of mRNA. Multiple forms of acetyl-CoA carboxylase (ACC) mRNA with 5'-end heterogeneity are generated as a result of differential splicing of two primary transcripts formed under the control of these two promoters. PI is responsible for the generation of class I mRNAs of ACC, which are induced in a tissue-specific manner under lipogenic conditions. PII generates class II mRNAs of ACC, which are expressed constitutively. Possible mechanisms for the regulation of PI under normal physiological conditions and agents that activate the promoter have been investigated. PI contains a TATA and a CCAAT box. In addition to these sequences, this promoter contains a 28-CA repeat sequence 220 bases upstream from the transcription initiation site; the presence of this sequence leads to about 70% repression of the basal promoter activity. Repression by the 28-CA repeat sequence requires the GCAAT sequence in the CCAAT box. The negative effect of the 28-CA repeat sequence is relieved by a CCAAT/enhancer-binding protein (C/EBP), which binds to the GCAAT sequence. Insertion of the 28-CA repeat sequence into the thymidine kinase promoter results in repression that can also be relieved by the C/EBP gene product. However, the same sequence exerts no effect on ACC promoter II, which has no CCAAT box. During the differentiation of 30A5 preadipocytes into adipocytes, the expression of class I ACC mRNA and C/EBP mRNA is coordinately increased. Therefore, the presence of the CA repeat in the promoter may be responsible for the inactivity of PI, and C/EBP may be one of the factors that is responsible for the activation of PI under lipogenic conditions. Interaction of the CA repeat and the CCAAT box in the repression and derepression of the ACC gene provides a novel function for the CCAAT box and C/EBP in gene regulation.

Cloning of human acetyl-CoA carboxylase cDNA.

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. In order to understand the mechanisms that regulate human acetyl-CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human acetyl-CoA-carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85% identity in nucleotide sequence with previously cloned rat acetyl-CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl-CoA-carboxylase mRNA species, which differ in the 5' untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type II acetyl-CoA-carboxylase mRNA contain 313- and 173-base-long 5' untranslated regions, respectively. The first 240 nucleotides in the 5' untranslated region of type I acetyl-CoA-carboxylase mRNA replace the first 100 nucleotides of the (G + C)-rich region of the 5' untranslated region of the type II mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue-specific manner.