Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.     

Water-jet dissection in renal surgery: experimental study of a new device in the pig

The pig model was used for experiments with a new type of water-jet dissector, which produces high-pressure water by application of a gas and maintains the water fully sterile in a single-use delivery apparatus. The experiment was conducted ex vivo (14 kidneys) and in vivo to compare electric cautery section with water-jet dissection (5 vs 11 partial nephrectomies). Ex vivo study confirmed sparing of blood vessels and pelvicaliceal system. In vivo study did not show significant differences in blood loss but, the water-jet allowed precise dissection and tight closure of the excretory system. More frequent haemorrhages were noted on histological examination of the WJ group, but no coagulation necrosis. These are preliminary findings and further studies of long-term results may confirm the benefits of the absence of parenchymal necrosis and the definite advantage of precise closure of the pelvicaliceal system, as morbidity of partial nephrectomy is often related to secondary haemorrhages and urinary fistulae.     

Effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial

BACKGROUND/AIMS: Silymarin has protective effects in different experimental conditions, but its efficacy in human liver cirrhosis has not been completely established. Therefore, this study was carried out to determine the effect of silymarin in alcoholics with liver cirrhosis with respect to survival and clinical and laboratory changes. METHODS: From February 1986 to June 1989, we enrolled 200 alcoholics with histologically or laparoscopically proven liver cirrhosis in a randomized, double-blind multicenter trial comparing 450 mg of silymarin (150 mg/ three times per day) with placebo. The primary outcome was time to death, and the secondary outcome was the progression of liver failure. Additional analyses were also performed in 75 patients in whom anti-hepatitis C virus antibodies were measured after completion of the trial. RESULTS: One hundred and three patients were assigned to receive silymarin and 97 to receive placebo. The two groups were well matched for demographic and baseline clinical and laboratory features. A 2-year study period was completed in 125 patients (57 receiving silymarin and 68 receiving placebo). Twenty-nine patients (15 receiving silymarin, and 14 receiving placebo) died during the trial. Survival was similar in patients receiving silymarin or placebo. The effect of silymarin on survival was not influenced by sex, the persistence of alcohol intake, the severity of liver dysfunction or by the presence of alcoholic hepatitis in the liver biopsy. Silymarin did not have any significant effect on the course of the disease. No relevant side-effects were observed in any group. CONCLUSIONS: The results of this study indicate that silymarin has no effect on survival and the clinical course in alcoholics with liver cirrhosis.     

Cystic thyroid lesions in children

BACKGROUND/PURPOSE: Cystic lesions of the thyroid encompass a wide and heterogeneous group of disease states in children, ranging from benign purely cystic entities to malignant tumors. The purpose of this study was to study both the presentation and management of cystic thyroid lesions in the pediatric population. METHODS: A retrospective review of all thyroid masses presenting between 1978 and 1996 and found to be purely or partially cystic on ultrasound examination was conducted, looking at presentation, family history, laboratory values, ultrasound scan and radionuclide imaging, and pathological and cytological evaluation. RESULTS: Twenty-four patients (19 girls, 5 boys) aged 6 to 18 years received the diagnosis of cystic lesions of the thyroid. Of these, 23 presented with painless neck masses, 21 were clinically euthyroid, only one had a single abnormal thyroid function test, only two had mildly positive antithyroid antibody test results, and nearly 30% had a positive family history of thyroid disease. Ultrasonography showed pure cysts in five patients and mixed solid cystic lesions in 19 patients. On scintiscan, six lesions were hot, 13 were cold, three showed normal uptake, and two were mixed. Treatment included either observation, aspiration, cyst sclerosis, surgery, or combinations thereof. Pathological and cytological results included follicular adenoma (n = 9), cystic degeneration (n = 6), multinodular goiter (n = 4), carcinoma (n = 2), branchial cleft cyst (n = 1), and undetermined (n = 2). CONCLUSIONS: Thyroid cysts are often thought to represent benign degenerative disease. Our study, which is the first in the literature to specifically address thyroid cysts in children, shows that ultrasound scan is useful in evaluating thyroid masses, whereas laboratory and radionuclide are of less value, and that single lesions of mixed echogeneity are likely to represent neoplasms, a significant percentage of which are malignant.     

The metabolism of phytanic acid and pristanic acid in man: a review

The branched-chain fatty acid phytanic acid is a constituent of the diet, present in diary products, meat and fish. Degradation of this fatty acid in the human body is preceded by activation to phytanoyl-CoA and starts with one cycle of alpha-oxidation. Intermediates in this pathway are 2-hydroxy-phytanoyl-CoA and pristanal; the product is pristanic acid. After activation, pristanic acid is degraded by peroxisomal beta-oxidation. Several disorders have been described in which phytanic acid accumulates, in some cases in combination with pristanic acid. In classical Refsum disease, the enzyme that converts phytanoyl-CoA into 2-hydroxyphytanoyl-CoA--phytanoyl-CoA hydroxylase--is deficient, resulting in highly elevated levels of phytanic acid in blood and tissues. Also in rhizomelic chondrodysplasia punctata, phytanic acid accumulates, owing to a deficiency in the peroxisomal import of proteins with a peroxisomal targeting sequence type 2. In patients affected with generalized peroxisomal disorders, degradation of both phytanic acid and pristanic acid is impaired owing to absence of functional peroxisomes. In bifunctional protein deficiency, the disturbed oxidation of pristanic acid results in elevated levels of this fatty acid and a secondary elevation of phytanic acid. In addition, several variant peroxisomal disorders with unknown aetiology have been described in which phytanic acid and/or pristanic acid accumulate. This review describes the discovery of phytanic acid and pristanic acid and the initial attempts to elucidate the origins and fates of these fatty acids. The current knowledge on the alpha-oxidation and beta-oxidation of these branched-chain fatty acids is summarized. The disorders in which phytanic acid and/or pristanic acid accumulate are described and some remarks are made on the pathogenic mechanisms of elevated levels of phytanic acid and pristanic acid.     

Nature of methamphetamine-induced rapid and reversible changes in dopamine transporters

The nature of methamphetamine-induced rapid and transient decreases in dopamine transporter activity was investigated. Regional specificity was demonstrated, since [3H]dopamine uptake was decreased in synaptosomes prepared from the striatum, but not nucleus accumbens, of methamphetamine-treated rats. Differences among effects on dopamine transporter activity and ligand binding were also observed, since a single methamphetamine administration decreased [3H]dopamine uptake without altering [3H]WIN35428 ([3H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate) binding in synaptosomes prepared 1 h after injection. Moreover, multiple methamphetamine injections caused a greater decrease in [3H]dopamine uptake than [3H]WIN35428 binding in synaptosomes prepared I h after dosing. Finally, decreases in [3H]dopamine uptake, but not [3H]WIN35428 binding, were partially reversed 24 h after multiple methamphetamine injections. Western blotting indicated that saline- and methamphetamine-affected dopamine transporters co-migrated on sodium dodecyl sulfate (SDS) gels at approximately 80 kDa, and that acute, methamphetamine-induced decreases in [3H]dopamine uptake were not due to loss of dopamine transporter protein. These findings demonstrate heretofore-uncharacterized features of the acute effect of methamphetamine on dopamine transporters.     

The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response

The nature of the initial interactions of African swine fever (ASF) virus with target cells is only partially known, and to date only the ASF virus protein p12 has been identified as a viral attachment protein. More recently, antibodies to viral proteins p54 and p30 have been shown to neutralize the virus, inhibiting virus binding and internalization, respectively. Therefore, we investigated the role of these proteins in the receptor-mediated ASF virus endocytosis in swine macrophages, the natural host cells. Proteins p54 and p30, released from ASF virus particles after treatment of virions with a nonionic detergent, bound to virus-sensitive alveolar pig macrophages. Binding of these proteins was found to be specifically inhibited by neutralizing antibodies obtained from a convalescent pig or from pigs immunized with recombinant p54 or p30 proteins. The baculovirus-expressed proteins p54 and p30 retained the same biological properties as the viral proteins, since they also bound specifically to these cells, and their binding was equally inhibited by neutralizing antibodies. Binding of 35S-labeled recombinant p54 and p30 proteins to macrophages was specifically competed by an excess of unlabeled p54 and p30, respectively. However, cross-binding inhibition was not observed, suggesting the existence of two different saturable binding sites for these proteins in the susceptible cells. In addition, protein p54 blocked the specific binding of virus particles to the macrophage, while protein p30 blocked virus internalization. Both proteins independently prevented virus infection and in a dose-dependent manner, suggesting that binding interactions mediated by both proteins are necessary to give rise to a productive infection. The relevance of blockade of virus-cell interactions mediated by p54 and p30 in the protective immune response against ASF virus was then investigated. Immunization of pigs with either recombinant p54 or p30 proteins induced neutralizing antibodies which, as expected, inhibited virus attachment or internalization, respectively. However, immunized pigs were not protected against lethal infection and the disease course was not modified in these animals. In contrast, immunization with a combination of p54 and p30 proteins simultaneously stimulated both virus neutralizing mechanisms and modified drastically the disease course, rendering a variable degree of protection ranging from a delay in the onset of the disease to complete protection against virus infection. In conclusion, the above results strongly suggest that proteins p54 and p30 mediate specific interactions between ASF virus and cellular receptors and that simultaneous interference with these two interactions has a complementary effect in antibody-mediated protection.     

Protective effect of exogenous nitric oxide on the renal function and inflammatory response in a model of ischemia-reperfusion

BACKGROUND: Tissue subjected to a period of ischemia undergoes morphological and functional damage that increases during the reperfusion phase. The aim of the present work was to assess the possible improvement induced by exogenous administration of nitric oxide (NO) on renal injury and inflammatory reaction in an experimental animal model of renal ischemia-reperfusion (I-R). METHODS: Ischemia was achieved by ligation of the left arteria and vein for 60 min, followed first by contralateral nephrectomy and then reestablishment of blood flow. Molsidomine, used as an NO donor, was administered by systemic injection 30 min before reperfusion. The effect of molsidomine was compared with the effect of hydralazine, a non-NO donor hypotensive agent. RESULTS: Treatment with molsidomine improved the renal dysfunction (increase in plasma creatinine and urea levels) caused by I-R. Moreover, molsidomine blunted the enhanced production of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin [IL] 1alpha), the increase in tissular levels of superoxide anions and oxygen free radical scavengers, and the neutrophilic infiltration observed in the ischemic kidney. One hundred percent survival was achieved in the group of animals treated with the NO donor, whereas the groups of animals undergoing I-R that did not receive molsidomine showed a 40% mortality from the second day after reperfusion. CONCLUSIONS: The present work demonstrated that systemic treatment with an NO donor before reperfusion improved renal function and diminished inflammatory responses in a kidney subjected to an I-R process.