The authors describe the development and the application of polymerase chain reaction (PCR) in the detection of hepatitis C virus. Based on the sequence data available in the literature, a conservative motif at the 5' end of the virus genome has been selected for amplification. Using the flanking synthetic oligonucleotides the specificity of the highly sensitive RNA-PCR assay was proved by serologically positive and negative serum samples. HCV-RNA-PCR studies were applied to start interferon treatment in patients from National Institute of Rheumatology and Physiotherapy (Budapest) with anti-HCV positive serum samples and this method was used to evaluate the efficacy of interferon therapy, as well.

Genome, Viral↗

PubMed1995

Expression of ril, a novel LIM domain gene, is down-regulated in Hras-transformed cells and restored in phenotypic revertants.

Several candidate genes involved in the maintenance of normal growth control (H-rev) were identified by differential expression cloning on the assumption that they are expressed in phenotypically normal rat cells and repressed in closely related H-ras transformed cells. Previously the genes coding for lysyl oxidase (H-rev142) and for an 18K-protein of unknown function (H-rev107) were recovered as cDNAs by subtraction cloning. Here we describe the identification and expression pattern of ril, a novel member of the heterogeneous group of genes encoding proteins with LIM/double zinc finger domains. The ril gene is expressed in normal fibroblasts and down-regulated in H-ras-transformed derivatives. Expression is restored in several independent phenotypic revertants derived from H-ras transformed cells. The predicted protein product of ril harbors a single LIM domain but lacks a homeodomain. The ril gene is highly conserved during evolution and is transcribed in various normal cell lines. Northern blot analysis and in situ hybridization studies showed that ril is expressed in meiotic spermatocytes, in somites of developing mice, and in a wide variety of tissues of adult mice.

Amino Acid Sequence↗

PubMed1995

Detection of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) (a note).

Since the majority of post-transfusion hepatitis cases are closely related to the HCV infections, the sensitive and specific detection of HCV-RNA is a central issue both in blood transfusion and in follow-up of hepatitis patients. This leaflet provides the technical details of HCV-RT-PCR.

Base Sequence↗

PubMed1994

HCV antibodies in Hungarian blood donations. Experiences collected by ELISA tests, immunoblot assays and polymerase chain reaction and protocols for donor management.

Routine screening of Hungarian blood donors for anti-HCV commenced in the second half of 1992. Before this, five available anti-HCV ELISA kits were compared in pilot studies. In the first series, 831 random donor samples were tested by one of the tests and the 12 (1.4%) reactives found were retested by the other four. Six of the reactives were positive in all ELISA. In the second series, 325 samples from donors with elevated transaminase levels were tested by all five kits. Forty-four were found to be reactive by one or more of the tests and 32 (10%) were positive in all five assays. Samples concordantly reactive in the ELISA were positive in second or third generation recombinant immunoblot assay (RIBA 2 or RIBA 3); those that gave discordant results were indeterminate or negative. Eleven concordantly reactive samples from the second series were HCV RNA positive by polymerase chain reaction (PCR). In the first period of screening with Abbott ELISA 2 a repeat-reactivity rate of 0.98% was observed in 171,106 samples tested. Reactives were retested for supplementary testing by Wellcozyme anti-HCV. Donors reactive in both tests and strongly reactive (ELISA ratio (ER) = optical density/cut off > or = 2.5) in either of them were permanently deferred. Those negative in the supplementary ELISA or weakly reactive (1.0 < or = ER < 2.5) in both tests were subjected to RIBA 2. On the basis of RIBA, positive donors were permanently deferred, indeterminates were excluded for 1 year and negatives were readmitted. In 1992, 1,347 supplementary tests were completed; 824 (61%) of the respective donors were permanently deferred, 218 (16%) were deferred for 1 year and 305 (23%) were readmitted.

Blood Donors↗

PubMed1994

Subtraction cloning of H-rev107, a gene specifically expressed in H-ras resistant fibroblasts.

We have isolated by subtractive hybridization a novel gene, called H-rev107, which is specifically expressed in a phenotypic revertant of H-ras transformed 208F rat fibroblasts. Apart from oncogene revertants, strong expression of H-rev107 was found in REF52 and EK-3 cells, two fibroblast lines resistant to transformation by activated H-ras oncogenes. In contrast, transformation-sensitive fibroblasts like 208F or NIH3T3 cells expressed only very little H-rev107 RNA. In H-ras or v-src transformed fibroblasts, H-rev107 RNA was undetectable. Introduction of the adenovirus E1A nuclear oncogene into ras-resistant REF52 cells abolished their transformation resistance and repressed the H-rev107 gene. H-rev107 encodes a protein with a molecular weight of 18 kDa without any structural similarity to known proteins. p18H-rev107 exists in two forms which can be distinguished by their electrophoretic mobility; one is localized predominantly in cell membranes, the other in the cytoplasm. In confluent contact-inhibited 208F cells, p18H-rev107 accumulated in cell membranes, while growth arrest induced by serum starvation did not induce H-rev107. In REF52, cell density had no influence on the expression or localization of p18H-rev107. Repression of the H-rev107 gene may be closely associated with the loss of density-dependent growth inhibition and with the expression of the neoplastic phenotype.

Adenovirus E1A Proteins↗

PubMed1993

Up-regulation of lysyl oxidase in spontaneous revertants of H-ras-transformed rat fibroblasts.

Neoplastic transformation mediated by ras oncogenes is associated with down-regulation of gene expression. We have constructed a subtracted complementary DNA library from preneoplastic rat 208F fibroblasts by hybridizing with mRNA from a ras-transformed subclone. One of the complementary DNA clones identified by this approach encodes the 3' end of lysyl oxidase, the homologue of the mouse ras recision gene. Expression of lysyl oxidase was almost completely down-regulated in two clones of H-ras-transformed 208F cells (FE-8 and FE-56). We isolated a set of spontaneous phenotypic revertants of FE-8 cells (designated FSR) by cloning at limiting dilution. FSR revertant clones expressed high levels of lysyl oxidase and H-ras mRNA but grew only poorly in semisolid agar medium as opposed to anchorage-independent parental FE-8 cells. We obtained subclones of FSR cells which displayed again the transformed morphology of FE-8 cells but required anchorage for growth and continued to express high levels of lysyl oxidase mRNA. Thus, expression of lysyl oxidase correlated with the suppression of anchorage-independent growth rather than with flat morphology. Lysyl oxidase might be a useful marker to distinguish between different aspects of reversion and transformation.

Algorithms↗

PubMed1993

Paradoxical sleep deprivatory effect of a single low dose of MPTP which did not produce dopaminergic cell loss.

In a previous study, we reported on a selective and long-lasting paradoxical sleep (PS) deprivation in cats following repeated administration of the Parkinson syndrome-inducing neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). While the characteristic motor deficits occurred only from the 2nd to 3rd day of a 5-day long administration of 5 mg/kg per day MPTP i.p., the PS deprivation started immediately after the first injection and lasted altogether for 11-13 days. The motor deficiencies induced by repeated administration of MPTP are mainly due to the selective depletion of dopaminergic neurons in the nigrostriatal system as the histological and biochemical data show. The immediate onset of PS deprivation and the PS recovery, despite the definite cell loss, suggests a mechanism independent of cell destruction. In our present study we investigated the occasional histological and the PS-deprivatory effect of a single low dose of MPTP in cats. A single injection of 2 mg/kg MPTP i.p. resulted in PS deprivation lasting for 2.5-3.5 h. The duration of other sleep stages showed no significant change and PS recovery was without rebound phenomenon, as in the case of repeated administration. Even a higher single dose of MPTP (5 mg/kg) resulted in no visibly detectable nigrostriatal cell loss. We suggest that the changes in monoamine release and/or turnover are involved in the PS deprivatory effect of MPTP.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1993

Suppression of ras-mediated transformation. Differential expression of genes encoding extracellular matrix proteins in normal, transformed and revertant cells.

A subtraction hybridization technique was used to identify genes specifically expressed in phenotypic revertants derived from cells transformed by the H-ras oncogene. The expression of genes coding for components of the extracellular matrix appears to be frequently down-regulated in transformed cells. Partial restoration is associated with the reexpression of the normal phenotype in revertants.

Animals↗

PubMed1992

[Reactivity of hepatitis C virus (HCV) antibody among blood donors in Hungary].

The anti-HCV results of 1958 donors are discussed. The Elisa positivity of high risk subjects was as follows: 60% in coagulopathies, 14% in transfused haematological patients and 32% in patients on dialysis. Elisa reactivity of random blood donors was 1.82%, in the group, selected on the basis of transaminase positivity it was over 8%. The confirmed results were compared by the commercially available Elisa tests. 30 positive samples were confirmed by RIBA or Abbott confirmation assay. 35 of the 55 anti-HCV positives were reactive by all the Elisa, 19 samples could be confirmed, 25 were not subjected to any confirmatory assay. Further studies, and the introduction of mandatory screening of blood donors for anti-HCV are necessary.

Blood Donors↗

PubMed1992

Stabilization of the oxy form of tyrosinase by a single conservative amino acid substitution.

Asp-208 of Streptomyces glaucescens tyrosinase (an invariant residue in the CuB-binding region of tyrosinases and haemocyanins) was conservatively substituted by glutamic acid. Although having little effect on spectroscopic or kinetic properties of the enzyme, the mutation greatly decreased the lability of Cu-bound O2. A rationalization for these results is given, based on the crystal structure of Panuliris interruptus haemocyanin in the conserved CuB-binding region.

Amino Acid Sequence↗

PubMed1992

Feeding disturbances and EEG activity changes after amygdaloid kainate lesions in the rat.

Kainic acid (KA), in various concentrations, was applied iontophoretically into the central nucleus of the amygdala. Microlesions with this cell specific neurotoxin caused body weight loss, hypo- or aphagia and hypo- or adipsia in a dose-dependent manner. EEG-examinations proved that even low doses of KA produced seizure activity; however, these epileptiform symptoms disappeared within the first 48 h after the operations. Thus, the lasting feeding disturbances produced by iontophoretic KA applications to the central nucleus of the amygdala (i.e., even these fine microlesions) were not related causally to the pathological EEG activity changes. Our findings, along with previous data, indicated that the body weight loss and feeding deficits were due to the KA-induced impairment of complex regulatory mechanisms.

Amygdala↗

PubMed1992

Microelectrophoretic application of kainic acid into the globus pallidus: disturbances in feeding behavior.

Body weight changes, food and water intake, and sensorimotor disturbances of male rats were studied after bilateral kainic acid-(KA) induced lesions of the globus pallidus (GP). To minimize the extent of damages, KA was applied electrophoretically by means of glass micropipettes (tip diameter of the pipettes was 10-15 microns). The neuron-specific damages of the GP resulted in aphagia and adipsia and rapid body weight decrease. Lesioned animals showed permanent motor disturbances but only temporary difficulties in the orientation toward sensory stimuli. Our data show that the selective destruction of the GP neurons results in a complex disorder that has motivational, (sensori)motor, and metabolic components.

Animals↗

PubMed1991

[Hepatitis C virus antibody in Hungarian blood donors, persons at risk and patients with post-transfusion hepatitis].

The authors screened blood donors, persons at high risk of infection and patients with post-transfusion hepatitis by the use of the new hepatitis C antibody test. 1.7% of persons donating blood acceptable for use according to current criteria were found positive. This ratio was markedly higher among donors displaying an elevated transaminase level. Among the various patients tested 85% of the haemophiliacs, 14% of the transfused haematology patients, and 40% of those on dialysis were found positive. In two groups of patients with post-transfusion non-A, non-B hepatitis the ratio of positives was 69% and 92%, respectively. The frequency of hepatitis C antibody was not sizably higher in the health personnel tested than that found in blood donors. The results indicate hepatitis C virus to be the main source of post-transfusion non-A, non-B hepatitis in Hungary too. As expected, HBc antibody revealing a previous contact with hepatitis B virus was found very frequent in patients with coagulopathies and in those on dialysis. A relatively high anti-HBc prevalence was found among health personnel and blood donors too. Authors intend to emphasize the need of thorough consideration of indications in hemotherapy although improvements in the screening of donations can diminish the risk of virus transmission by blood or blood derivatives.

Blood Donors↗