Use of phospholipids to repair rat liver membranes during carbon tetrachloride poisoning

The effect of phospholipid multilayer liposomes on the properties of endoplasmic reticular membranes has been studied in hepatic cells damaged with CCl4. It has been shown that the repair effect of phosphatidylcholine liposomes was manifested in vivo by normalization of phospholipid membrane composition, reduction in the degree of cytochrome P-450 inactivation, partial normalization of its hydroxylase activity and recovery of glucose-6-phosphatase activity. The degree of phosphatidylcholine unsaturation, and the introduction of antioxidants--SH-compounds, sphingomyelin, phosphatidyl inositol--into liposomes did not influence the efficacy of liposomes.     

Metabolic requirements for interactions between nuclear and cytoplasmic membranes in the repair of damaged Amoeba nuclei.

Amoeba nuclear envelopes were damaged using microsurgery, and metabolic requirements for the steps in their repair were studied, and my placing the cells in a solution containing one of several metabolic inhibitors. The first step in repair, the association of pieces of endoplasmic reticulum with holes in the nuclear membranes, appears to be a passive process since it was not affected by inhibitors of energy production, RNA synthesis, or protein synthesis. In contrast, fusion of pieces of endoplasmic reticulum with the nuclear membranes at the margins of the holes was blocked by KCN and dinitrophenol, indicating that membrane fusion requires energy derived from respiration, but RNA and protein synthesis inhibitors did not prevent fusion of pieces of endoplasmic reticulum with the nuclear membranes. The subsequent completion of repair and restoration of intact nuclear membranes was almost completely blocked by inhibitors of respiration, and it was reduced in the presence of actinomycin and emetine, suggesting that in addition to a requirement for energy, some later steps in the repair of the nuclear membranes require RNA and protein synthesis.     

Enzymes involved in the repair of damaged DNA

The multitude of enzymes responsible for removing damaged nucleotides from DNA in an error-free manner is reviewed. The most direct mechanisms include enzymatically catalyzed photoreversal of cyclobutane dimers and the removal of the O⁶-methylguanine adduct from alkylated DNA by an enzyme whose presence is dependent on adaptation. The direct removal of either damaged purines or pyrimidines or partial removal of photochemically induced diadducts is catalyzed by DNA glycosylases in the absence of phosphodiester bond hydrolysis. Incision of DNA containing apurinic or apyrimidinic sites arising either spontaneously or by the action of DNA glycosylases is catalyzed by specific endonucleases. The incision of DNA containing bulky adducts is attributed to a multigenically controlled uvr system in Escherichia coli. The mechanisms of damaged nucleotide excision and reinsertion of nucleotides are controlled by unique exonuclease functions in either direct or indirect association with DNA polymerases.     

Interactions between endoplasmic reticulum and nuclear membranes of different types of cells during repair of damaged Amoeba nuclei

Repair of amoeba nuclear envelopes that have been damaged microsurgically involves the association of pieces of endoplasmic reticulum with the damaged nuclear membranes. The capacity of endoplasmic reticulum of one type of cell to interact with the nuclear membranes of a different type was tested by placing the damaged nucleus of one kind of amoeba into the cytoplasm of another. Damaged nuclei from Amoeba proteus underwent repair in the cytoplasm of A. discoides or A. indica, as was the case in the reciprocal combinations of these nuclei and cytoplasms. In samples prepared 30 min after operation, heterologous endoplasmic reticulum was associated with holes in the nuclear membranes and appeared to fuse with the nuclear membranes at the margins of the holes. By 5 h after operation, almost all of the cells survived, and the nuclear membranes were largely intact, indicating that repair had occurred. In contrast, when an Amoeba dubia nucleus was damaged and placed in A. proteus cytoplasm there was no evidence of repair and many cells died within a few hours. The results indicate that endoplasmic reticulum and nuclear membranes from different types of cells can interact during repair of damaged nuclear membranes. There appears to be a specificity to this interaction, however, since in a combination of relatively dissimilar cells no association of endoplasmic reticulum with damaged nuclear envelopes was observed and repair did not occur.     

Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes.

A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.     

Recombinational repair and restart of damaged replication forks

Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next.     

Oxidatively damaged DNA and its repair in colon carcinogenesis

Inflammation, high fat, high red meat and low fiber consumption have for long been known as the most important etiological factors of sporadic colorectal cancers (CRC). Colon cancer originates from neoplastic transformation in a single layer of epithelial cells occupying colonic crypts, in which migration and apoptosis program becomes disrupted. This results in the formation of polyps and metastatic cancers. Mutational program in sporadic cancers involves APC gene, in which mutations occur most abundantly in the early phase of the process. This is followed by mutations in RAS, TP53, and other genes. Progression of carcinogenic process in the colon is accompanied by augmentation of the oxidative stress, which manifests in the increased level of oxidatively damaged DNA both in the colon epithelium, and in blood leukocytes and urine, already at the earliest stages of disease development. Defence mechanisms are deregulated in CRC patients: (i) antioxidative vitamins level in blood plasma declines with the development of disease; (ii) mRNA level of base excision repair enzymes in blood leukocytes of CRC patients is significantly increased; however, excision rate is regulated separately, being increased for 8-oxoGua, while decreased for lipid peroxidation derived ethenoadducts, ?Ade and ?Cyt; (iii) excision rate of ?Ade and ?Cyt in colon tumors is significantly increased in comparison to asymptomatic colon margin, and ethenoadducts level is decreased. This review highlights mechanisms underlying such deregulation, which is the driving force to colon carcinogenesis.     

Ionic permeability of sarcoplasmic reticulum membranes damaged by x-rays

As early as 1 and 24 h following single local X-irradiation (0.21 C/kg) of rabbit hindlimbs an increase was noted in the permeability of skeletal muscle sarcoplasmic reticulum membranes for Ca2+, K+ and Na+. The effect was maximum 1 h after irradiation and more pronounced for K+ and Na+ than Ca2+.     

Decreased repair of gamma ray damaged DNA in progeria.

A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced in the progeria lines as compared to normal fibroblasts when gamma-irradiated virus was used, indicating a defect in the repair of gamma ray damaged DNA in the progeria cells.     

Labeling of glucagon binding components in hepatocyte plasma membranes.

A photosensitive derivative of glucagon, ¹²⁵I-N^?-4-azido-2-nitrophenyl-glucagon, has been synthesized and used to specifically label glucagon binding proteins in hepatocyte plasma membranes. Photolysis of the derivative in the presence of a membrane suspension results in the incorporation of radioactivity primarily into membrane components with a molecular weight range of 23,000–25,000. The binding properties of the derivative are essentially identical to that observed for glucagon. The binding of ¹²⁵I-NAP-glucagon was completely inhibited in the presence of glucagon (3 μM) while greater than 90% of the covalent labeling was also inhibited in the presence of glucagon. These studies suggest that the labeled membrane protein may be a component of the glucagon receptor.     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子(rhHGF)对CCl4染毒肝细胞的保护作用. 结果表明: (1) rhHGF (5 ng/ml)预处理后可显著提高CCl4 (15 mmol/L)染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶(ALT)、 K+的漏出;(2) 表皮生长因子(EGF,50ng/ml)和rhHGF (5 ng/ml)合用预处理肝细胞,CCl4染毒后细胞内ALT、 K+漏出较rhHGF和EGF单独保护组进一步降低;(3)大鼠肝部分切除和CCl4 (50%,2.5 ml/kg bw)染毒后,再生肝内HGF基因及其受体基因/c-met的表达分别较假手术和盐水对照组显著升高. 结果提示,rhHGF对CCl4染毒大鼠肝细胞具有保护作用;EGF和rhHGF有协同保护作用;HGF及其受体的表达在肝脏再生及修复中可能有重要作用.     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子（ｒｈＨＧＦ）对ＣＣｌ４染毒肝细胞的保护作用。结果表明：（１）ｒｈＨＧＦ（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／Ｌ）染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶（ＡＬＴ）、Ｋ＾＋的漏出;（２）表皮生长因子（ＥＧＦ,５０ｎｇ／ｍｌ）和ｒｈＨＧＦ（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

The activation of factor X by hepatocyte plasma membranes

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.     

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes an increase in protein tyrosine kinase activities at an early stage of poisoning in vivo in rat hepatocyte membranes

TCDD was found to cause a significant rise in protein tyrosine kinase levels at an early stage of poisoning in rat liver membrane preparations. The results of sephadex G-150 column chromatographic analysis on rat hepatocyte membranes indicate that there are at least three tyrosine kinases of which activities increase as a consequence of TCDD treatment in vivo. The TCDD-evoked rise in such protein tyrosine kinase activity precedes the down-regulation of the EGF (epidermal growth factor) receptor in the plasma membrane in vivo.     

Ornithine decarboxylase: a biochemical marker of repair in damaged tissue

The purpose of this study was to: (a) determine the temporal pattern of expression of ornithine decarboxylase (ODC), an established marker of cells engaged in proliferation and differentiation, during repair of traumatized skeletal muscle, and (b) evaluate ODC as a biochemical marker for indexing the extent and rate of repair of traumatized skeletal muscle in response to therapeutic agents. Adult female Wistar rats weighing 240 to 280 grams were anesthetized and injected with 100 ul of 2% lidocaine into the right or left anterior tibialis muscle to induce a localized injury. The animals were treated 1 hour post-injection and every 12 hours for five minutes up to the time of sacrifice using ultrasonic irradiation at 1.5 watts/cm2, sham-irradiation, or no treatment. An analysis of variance for repeated measures and a Tukey's post hoc test were used to determine the significance of treatment effects. Animals irradiated with ultrasound demonstrated an accelerated pattern of change in ODC activity at 24 hours (P less than 0.001), 30 hours (P less than 0.003), and at 48 hours (P less than 0.002) compared to control and sham-irradiated animals at these time intervals. Irradiated animals also demonstrated less edema at 48 hours compared to sham-irradiated and control animals (P less than 0.003). These findings suggest that ODC is a useful biochemical marker for determining the extent and rate of tissue repair in traumatized skeletal muscle, and it provides a temporal and quantifiable parameter for evaluating the efficacy of therapeutic agents used to treat damaged skeletal muscle.