Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of γ-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed γ-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.     

Inhibition of a viral prolyl hydroxylase

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

An increase of collagen biosynthesis in livers of rats submitted to chronic intoxication with ethanol

Summary It was found that chronic intoxication of rats with ethanol results in a dictinct, progressive increase of 5³H-proline incorporation into collagen synthesized by liver. At the same time biosynthesis of other proline-containing proteins in rat liver slightly decreases. These effects precede the morphological symptoms of liver damage induced by ethanol.     

Modulation of biosynthesis of phosphatidylcholine via CDP-choline in rat liver: Influence of ethanol on the microsomal cholinephosphotransferase activity

We have studied in vitro the effects of ethanol on the different enzymes involved in the biosynthesis of phosphatidylcholine (PC) via CDP-choline. Ethanol alters neither choline kinase (CK) nor CTP:phosphocholine cytidylyltransferase (CT) activities but, at levels higher than 50 mM, it does significantly inhibit microsomal cholinephosphotransferase (CPT) activity concomitantly with an increase in the ethanol concentration. A study of the kinetics of the reaction catalysed by CPT shows that ethanol decreases V_max without altering K_m, indicating a non-competitive inhibitory effect. An analysis of the thermodependence of CPT activity in the absence of ethanol reveals a break in the Arrhenius plot and thus a straight relationship between enzyme activity and the physico-chemical state of the microsomal membrane. Incubation of microsomes in the presence of ethanol increased the transition temperature from 25.8–28.2°C. Microsomes were also incubated with n-alkanols with chain-lengths of fewer than five carbon atoms at concentrations which, according to their partition coefficients, produce equimolar levels in the membrane. Under these conditions all the alkanols caused the same inhibitory effect. All these results demonstrate that ethanol modulate the PC biosynthesis at the level of CPT activity and does not affect the CT enzyme. The inhibition found on CPT is clearly dependent on the alteration produced by ethanol on the hepatic microsomal membrane.     

延龄草对LPS诱导大鼠肝损伤的保护作用及机制

目的：研究延龄草（TTM）对脂多糖（LPS）诱导大鼠氧化应激与肝损伤的保护作用。方法：SD大鼠60只,按体重随机分成TTM高、中、低剂量组、模型组、地塞米松磷酸钠（DEX）对照组及空白对照组（n=10）。TTM高、中及低剂量组按（8、4、2） g/（kg·d） TTM灌胃,模型组、DEX对照组及空白对照组灌胃等量蒸馏水,每隔5 d,TTM高、中、低剂量组、模型组、DEX对照组按1 mg/kg腹腔注射LPS,DEX对照组同时腹腔注射DEX（5 mg/kg）,空白对照组注射等量生理盐水。30 d后,测定大鼠胸腺指数、脾脏指数,对血清一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）活性与一氧化氮（NO）、谷胱甘肽（GSH）、硫代巴比妥酸反应产物（TBARS）、白细胞介素6（IL-6）、IL-10及肿瘤坏死因子α（TNF-α）含量,肝组织SOD、谷胱甘肽过氧化氢酶（GSH-Px）活性与GSH、TBARS含量进行检测。结果：与模型组相比,TTM高剂量组在（19~30） d体重显著降低（P<0.05）,TTM高、中、低剂量组胸腺指数,TTM高剂量组脾脏指数显著降低（P<0.05）,TTM高、中、低剂量组血清NOS活性与TBARS、NO含量显著降低（P<0.05）,TTM高剂量组血清SOD活性及中、高剂量组GSH含量显著上升（P<0.05）,TTM高、中剂量组血清IL-6、TNF-α含量显著降低,IL-10含量显著升高（P<0.05）,TTM中、高剂量组肝脏TBARS含量显著降低,TTM各剂量组肝脏SOD活性与中、高剂量组GSH-Px活性,高剂量组GSH含量显著升高（P<0.05）。结论：TTM对LPS所致大鼠的胸腺、脾脏萎缩有一定的延缓作用,能有效降低血清中NOS活性,减少NO生成,提升SOD、GSH-Px活性与GSH含量,减轻脂质过氧化,降低IL-6、TNF-α过量分泌、提升IL-10含量,有抗炎护肝的功能。     

Histological changes in the liver of fetuses of alcohol-treated pregnant rats

In this study, immunolocalization of tenascin (TN), type IV collagen and histopathological changes in the liver of fetuses of pregnant Wistar albino rats treated with two doses of alcohol (1 and 5 g kg(-1) day(-1)) were determined. The samples of liver obtained from fetuses of rats on day 21 of gestation were evaluated morphologically and fixed for histology and immunohistochemistry. The sections stained with H&E and peroxidase-antiperoxidase (PAP) method for TN and type IV collagen were evaluated and immunohistochemical staining results were examined by the intensity of labelling. Statistical evaluation of data showed that there were no differences in liver weights between the control and calorie control groups; but some differences in relative liver weight between the control and alcohol treatment groups were determined. The changes in the liver histology such as degeneration of the cytoplasm of hepatocytes and increases in the number of megakaryocytes were seen in the fetuses of groups receiving alcohol during pregnancy. Increases in the immunolocalization of TN and type IV collagen in the liver of fetuses of alcohol-receiving rats were also found.     

Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase

The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. -butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of -butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

The protective effects of caffeic acid phenethyl ester (CAPE) against liver damage induced by cigarette smoke inhalation in rats

The aim of this study was to investigate the histological and biochemical changes in liver of rats exposed to cigarette smoke and effects of caffeic acid phenetyl ester (CAPE) on these changes. For this purpose, 21 male Wistar rats were divided into three groups. Animals in Group I were used as control. Rats in Group II were exposed to cigarette smoke and rats in Group III were exposed to cigarette smoke and injected daily with CAPE. At the end of the 60-days experimental period, all rats were killed by decapitation and blood samples were obtained. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin levels and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px ), malondialdehyde (MDA) contents were determined. Following routine histological procedures, liver tissue specimens were examined under a light microscope. The levels of ALT, AST, total bilirubin, SOD, GSH-Px and MDA were significantly increased in rats exposed to cigarette smoke compared with those of the controls. Light microscopic examination of liver specimens from rats exposed to cigarette smoke revealed mononuclear cell infiltration and that some of the hepatocytes had a hyperchromatic nucleus and enlarged sinusoids. The rats which were treated with CAPE along with cigarettes had partially attenuated histological changes associated with cigarette exposure. In conclusion, the damage inflicted by cigarette in the rat liver can be partially prevented by CAPE administration.     

Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis

Summary Steatohepatitis has recently been increasing as a cofactor influencing the progression of fibrosis, cirrhosis, adenoma and carcinoma in liver; however, the mechanisms by which it contributes to liver injury remain uncertain. We induced steatohepatitis in zebrafish embryos using thioacetamide (TAA). TUNEL assay revealed significant increasing of apoptosis in liver after 5 days post fertilization and the increasing of apoptosis was observed to be associated with the up-regulation of apoptotic genes such as, bad, bax, P-38a, caspase-3 and 8, and JNK-1. Histological sections by oil red O stain showed the accumulation of fatty droplets which causes the pushing of the nucleus towards one side. Up-regulation of steatosis markers such as, ACC, adiponectin, PTL, CEBP- and , SREBP-1 was also observed. Furthermore, the elevation of glutathione peroxidase in TAA treated embryos indicated that TAA induces lipid peroxidation which leads to causes liver damage. Zebrafish has already been considered as a good human disease model and in this context; TAA-treated zebrafish may serve as a good animal model to study the molecular pathogenesis of steatohepatitis. Moreover, non-availability of specific drugs to prevent steatohepatitis, this animal model may serve as a powerful preclinical platform to study the therapeutic strategies and for evaluating chemoprevention strategies for this disease.     

TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain

New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of β2i, β5i and PA28α, although it decreases the 20S constitutive proteasome subunits (α2, β5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage.     

Leucocytosis as a marker of organ damage induced by chronic strenuous physical exercise

The effects of strenuous physical exercise on the serial changes in the haematological, biochemical and hormonal markers were investigated. A group of 14 soldiers, aged 24–36 years, took part in a military training course for about 13 weeks. After severe exercise stress, an increase (90%) in the number of peripheral blood leucocytes was observed. The degree of leucocytosis showed a close correlation with the values of some serum parameters, such as concentrations of aspartate aminotransferase (AST;r = 0.747), lactate dehydrogenase (LD;r = 0.748), blood urea nitrogen (r = 0.756), creatine kinase (CK;r = 0.637), manganese-superoxide dismutase (Mn-SOD;r = 0.508), alanine aminotransferase (ALT;r = 0.542) and uric acid (r = 0.538), and concentrations of urinary parameters, such as vanilmandelic acid (r = 0.429) and free cortisol (r = 0.437). The subjects showing prominent leucocytosis over 9500 cells · l^–1 exhibited a lower concentration of serum cholinesterase than those who showed milder leucocytosis. The serum Mn-SOD concentration was closely correlated with the serial changes in serum concentrations of AST, ALT, LD and CK, indicating exercise-induced muscle and liver damage. The change in peripheral leucocyte number was assumed to be diagnostically informative and may be a prognostic marker, reflecting organ damage and restoration after strenuous physical exercise.     

Change of tyrosine hydroxylase in the parkinsonian brain and in the brain of MPTP-treated mice as revealed by homospecific activity

Changes in homospecific activity (unit of enzyme activity per unit of enzyme protein; Rush, Kindler and Udenfriend, 1974. Biochem. Biophys. Res. Commun., 61, 38) of tyrosine hydroxylase (TH) in the striatum of the brain were examined in MPTP-treated mice and parkinsonian patients. After a single injection of MPTP to mice, TH activity was acutely inhibited onlyin situ without changes in in vitro TH activity (Vmax) and TH protein; TH homospecific activity (TH Vmax/TH protein) did not change. After repeated injection of MPTP to mice for 8 days, in situ TH activity, in vitro TH Vmax, and TH protein were decreased in parallel, and TH homospecific activity did not change The result indicates that the decreases in in situ TH activity and in TH Vmax are due to the decrease in TH protein by nerve degeneration of dopaminergic neurons in MPTP treated mice. However, when MPP⁺ was infused in the striatum of rats for 3 hours, in vitro TH activity (Vmax) was decreased without changes in TH protein. Thus, TH homospecific activity was decreased. The results indicate that MPP⁺ inactivates TH protein in the striatum after continued infusion. In contrast, the homospecific activity of TH in post-mortem parkinsonian striatum was increased 3-fold. The increase in homospecific activity of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.Special issue dedicated to Dr. Sidney Udenfriend.     

Immunohistochemical evidence for the presence of tryptophan hydroxylase in the brains of insects as revealed by sheep anti-tryptophan hydroxylase polyclonal antibody

Immediately following the discovery of tryptophan hydroxylase in Drosophila, we demonstrated the presence of tryptophan hydroxylase in the brain of the beetle Harmonia axyridis (Coleoptera: Coccinellidae). However, whether tryptophan hydroxylase is present in the brains of other insects is still a matter of discussion. In the current study, sheep anti-tryptophan hydroxylase polyclonal antibody has been applied to test for tryptophan hydroxylase immunoreactivity in a broader taxonomic range of insect brains, including holometabolous and hemimetabolous insects: one species each of Coleoptera, Hymenoptera, Diptera, and Blattaria, and two species of Lepidoptera. All species show consistent tryptophan hydroxylase immunoreactivity with distribution patterns matching that of serotonin. The immuno-positive results of such an antibody in brains from diverse orders of insects suggest that specific tryptophan hydroxylase responsible for central serotonin synthesis is probably present in the brains of all insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from the National Natural Science Foundation of China (grant no. 30470546) and the Natural Science Foundation of Jilin Province (grant no. 20030550–7).     

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.     

Levels of collagenolytic activity, β-glucuronidase,and collagen prolyl hydroxylase in paws from rats with developing adjuvant arthritis

The collagenolytic activity associated with insoluble collagen fibers separated from homogenates of inflamed paws from rats with adjuvant arthritis was quantitated using EDTA-sensitive solubilization of hydroxyproline as a measure of activity. Approximately 60% of the solubilized hydroxyproline was associated with dialyzable products. The level of collagenolytic activity in the paws increased with time after the induction of adjuvant arthritis and paralleled to a large extent the development of inflammation in both the adjuvant injected (right) hind paw and in the non-injected, contralateral paw. By day 26, the level of free collagenolytic activity in the injected paw had increased to a level 30 times normal while that in the contralateral paw had increased to a level 10 times normal. Treatment of the residues from the injected paws with trypsin resulted in the activation of a latent collagenolytic activity which, on day 26, accounted for approximately 50% of the total activity. The elevated level of collagen prolyl hydroxylase in the inflamed paw suggested that the rate of collagen synthesis was also increased. The activity of β-glucuronidase increased in the inflamed paw with time after the induction of adjuvant arthritis while that of cathepsin G was elevated as compared to normal in paws removed, 5 but not 22 days after the induction of adjuvant arthritis. The inflamed paw of the adjuvant rat may represent a useful system in which to study the role of collagenolytic enzymes in the destruction of connective tissue by inflammatory lesions.