One gene, two transcripts: isolation of an alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary Sjögrens' syndrome.

A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjögrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear autoantigen La/SS-B. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF-kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear autoantigen La/SS-B alters in dependence on disease conditions.

Tissue and age specific expression of the myc proto-oncogene family throughout the life span of the C57BL/6J mouse strain.

The expression of the proto-oncogene myc family (c, L and N) in terms of steady-state mRNA levels was determined in seven different normal non-cancerous tissues throughout the life span (seven different ages) of the C57BL/6J male mouse strain. C-myc oncogene expression was highest in prenatal and newborn ages and then decreased to its lowest levels at about 6 months of age. With further increase of age, a progressive pattern of increase in expression of c-myc was found in brain, liver, skin, and small intestine. However, for kidney, spleen and heart, little or no significant change was evident. Significant differential expression of c-myc was found in most tissues in animals of the same age, with highest expression consistently being found in spleen and liver at all ages. For the N-myc and L-myc oncogenes, expression was also highest in prenatal and newborn tissue as compared to the 6-month young adult, but little or no further change was found at older ages. However, substantial tissue-dependent differences in expression were also found, and no expression at all was detected at any age for N-myc in liver and for L-myc in heart, small intestine and liver. Taken together, these results indicate that the expression of the proto-oncogenes c-, L- and N-myc is dependent not only on tissue and embryonic development, as previously shown by other workers, but also on age past the young adult stage of life span. The age-dependent increase in expression of c-myc oncogene found in normal-appearing non-cancerous tissues is of particular interest as possibly reflecting tissue alterations related to both the aging process and the age-dependent increase in cancer incidence.

Analysis of expression of the gene encoding for the nuclear autoantigen La/SS-B using reporter gene constructs.

In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was replaced with the exon 1'. Moreover, exon 1' La mRNAs were found to start at different 5'-regions. In dependence on the 5'-start the exon 1' La mRNAs encoded for up to three open reading frames upstream of the La frame, which starts in the exon 2. The exon 1' was located in the intron about 70 nts downstream of the exon 1. The exon 1' La mRNA was proposed to be the result of a promoter switch in combination with an alternative splicing mechanism. The commonly used technique to study the expression of a eucaryotic gene is to fuse a reportergene immediately downstream of the proposed regulatory elements. Due to (i) the short distance between exon 1 and exon 1', (ii) the varying 5'-starts of the exon 1' La mRNAs, and (iii) the upstream open reading frames in the exon 1' La mRNAs this technique appeared to be difficult to apply to the La gene. In order to overcome these problems a luciferase reportergene construct was cloned which started about 2500 nts upstream of the exon 1 and contained the exon 1, the intron including the exon 1', and a portion of the exon 2. Luciferase was fused into the exon 2. This construct was used to prepare 5'-deletion mutants. The constructs were transiently transfected into HeLa cells. RNAs were isolated from the transiently transfected cells and analyzed using the 5'-Rapid Amplification of cDNA End technique. The PCR products were subcloned and sequenced. This analysis showed that exon 1 and exon 1' transcripts were correctly transcribed and spliced from the La luciferase fusion construct. Moreover, the 5'-start of the respective transcript allowed to identify those genomic regions in the La gene that were most likely being involved in determining the respective transcription initiation site. In parallel to the estimation of the 5'-start of the transcripts, the luciferase activity was measured. Thereby we detected a cryptic promoter element in the intron between the exon 1 and exon 2.

Isolation of rat cDNA clones coding for the autoantigen SS-B/La: detection of species-specific variations.

Clones of cDNA coding for the autoantigen La (or SS-B) were isolated from a library made from rat liver. A comparison of the rat La cDNA (encoding from nt 38 to 1281 for rat La protein) with the sequences known for human and bovine La protein resulted in the identification of species-specific inserts. The inserts seem to be the result of multiplication of flanking sequences during evolution. In addition to these variations, we observed that rat La cDNAs exhibit non-canonical polyadenylation sites. Finally, a databank search resulted in the identification of a DNA sequence originally termed as TAG or TSG20X (GenBank accession No. X61893) which represents the C terminus of mouse La/SS-B protein.

On the nature of aging.

Most of the aging theories are monistic in nature, they omit numerous key factors of senescence during the process of model creation. There are two main categories of these theories: program theories and error (mutation) ones. Program theories imply the existence of internal or external programs that determine the aging process ab ovo. The error theories involve explicit or implicit the idea that aging would not happen without the destructive factors that cause errors, mutations, regulation disorders, and in turn these processes finally lead to disfunctions and senescence. The aim of this paper is to indicate that aging may be multifactorial and the process of senescence may be determined by the information level of the organization. This level itself changes during senescence (including the information level of the genom that also alters by time because of, e.g. its 'fluid' character). According to this approach the aging process is determined by the sum effects of internal (e.g. genom) and external (material, energy, information) factors, although there are some elements that bear more importance than others. Subsequently, the maximal life-span is probably determined by the principle of the weakest element of the chain. Because of the high complexity of the human body where different information systems superpose each other, the cooperation of the elements (counter-effects, regulation) have the same determining importance as the information level of the unit parts (cells) have. The further aim of this paper is to show that the roots of certain diseases (e.g. cancer) could firmly be linked to the aging process itself. This interpretation offers two ways of influencing the process of senescence. It could be influenced by maintaining the information level of the organism via optimization or by changing (elevating) this level. All the factors that help to prevent the decrease of the information level of the organism could act against aging and certain diseases, and vice versa: the factors which deteriorate the state of the information system could contribute to the acceleration of the aging process.

Expression of mRNAs of pancreatic and L type RNase inhibitors as a function of age in different tissues of SAMP8 and BDF1 mice.

Turnover of mRNAs could be influenced not only by the synthesis of different mRNA species but also by the altered levels of mRNA-degrading enzymes such as RNases and their endogenous inhibitors. In the present work we evaluated possible age-related changes in the mRNA levels of pancreatic as well as L type RNase inhibitors in five different tissues of the BDF1, SAMR1 and SAMP8 using Northern blots. The mRNA levels varied depending on the tissues and mouse strains studied. In certain instances such as the RNase L inhibitor mRNA levels in the lung of SAMP8, there was a statistically significant (P < 0.05) increase of 40% if we compared the young (3 months old) and old (18 months old) animals. These changes could possibly contribute to a certain extent to the already lower levels of mRNAs due to decreased transcriptional activities in aged animals.

Expression of superoxide dismutase and catalase in rat brain as a function of age.

Active oxygen species have been proposed to be involved in the aging process of the brain, therefore alterations of the levels of enzymes involved in the defence system against free radicals and other active species could substantially influence the aging process. In this study the enzyme activities of superoxide dismutase (Cu/Zn) and catalase as well as the relative levels of their mRNA were measured in the brain of Fischer F344 rats of various ages (5-37 months old). A gradual decrease in the activity of these enzymes (21-27%) was observed with increasing age. The alterations were paralleled by a decrease (39-40%) in the relative levels of these mRNA species. Thus the decrease in the activity of superoxide dismutase and catalase appears to be due to an age-dependent change in the expression of these genes.

Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats: an explanation of its action in terms of the membrane hypothesis of aging.

The rates of total and polyA+ RNA (mRNA) synthesis were measured by radioisotope technique in the brain cortex of female CFY rats. There was practically no significant difference between the young (1.5 months) and adult (13 months) rats; however, the old group (26 months) displayed a considerable decrease of the rates of synthesis of both classes of RNA studied. Centrophenoxine treatment (100 mg per kg body weight per day, for 2 months) reversed this tendency, and increased significantly the synthesis rates of old rats almost to the adult level. The results are interpreted in terms of the membrane hypothesis of aging, attributing a free-radical scavenger function of the dimethylamino-ethanol incorporated into the nerve cell membrane from the centrophenoxine.

Dysdifferentiation hypothesis of aging and cancer: a comparison with the membrane hypothesis of aging.

Our laboratories have been testing the basic concept that the age-dependent deterioration of the molecular components of living systems may be due in part to the biochemical effects of active oxygen species. The dysdifferentiation hypothesis of aging and cancer (DHAC) as well as the membrane hypothesis of aging (MHA) are discussed and compared to each other. These two hypotheses consider cellular mechanisms through which free radical-induced alterations may lead to the aging process. DHAC emphasizes the importance of the instability of the differentiated state of cells and how active oxygen species may interact with the genetic apparatus of cells, leading to improper gene regulation. The evidence supporting this hypothesis includes an age-dependent increase in the expression of specific genes that normally are expected to be repressed. Such evidence now includes the c-myc oncogene as well as an age-dependent decrease in the average methylation level of the entire genome in liver tissue of mice. The central concept of DHAC is that aging is a result of gene regulatory instability and that lifespan is governed by mechanisms acting to stabilize proper gene regulation. MHA is based on the concept that all cellular components are exposed to free-radical attacks, and that the damaging efficiency of the radicals is density-dependent. Compact structures like membranes are consequently more susceptible to damage than cytosolic components. In addition, the cell plasma membrane is exposed to another damaging effect called residual heat damage, which is due to the depolarization-induced discharge of the membrane during the action potential. MHA predicts that a key process of normal differentiation as well as aging is a continuous, age-dependent loss of the passive permeability of the cell membrane for potassium and probably also for water. This is due to a constant difference between the rates of damage and replacement of the membrane components and results in a gradual dehydration of the intracellular mass from the embryonic state to the aging state. The increasing intracellular density will eventually become rate-limiting for many different cellular functions, resulting in the cessation of growth and the beginning of aging. MHA also predicts an overall decrease of gene expression and protein turnover rate during aging. Pharmacological interventions on the cell membrane have supported the validity of MHA and have indicated specific mechanisms of how aging and dysdifferentiation may occur.

UVB light and 17-beta-estradiol have different effects on the mRNA expression of Ro/SSA and La/SSB autoantigens in HaCaT cells.

Antibodies produced against the Ro/SSA and La/SSB autoantigens are not only of diagnostic value but they may even play a role in the pathogenesis of several autoimmune diseases (Sjögren's syndrome, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus and systemic lupus erythematosus). Among other factors, ultraviolet (UV) radiation and also the hormonal milieu are well-known cofactors in the pathogenesis of these autoimmune diseases. The goal of our research was to study the possible alterations in mRNA levels of three different Ro antigens and that of two La species produced by alternative splicing in transformed human keratinocytes (HaCaT cells) after UVB irradiation and after 17-beta-estradiol treatment. The polymerase chain reaction technique was used to determine the mRNA levels of the Ro and La species after 24, 48, and 72 h of irradiation. The mRNA levels of calreticulin increased as a function of time after UV irradiation but the mRNA levels of 52 kDa and 60 kDa Ro mRNAs were unaltered. After treating the cells with 17-beta-estradiol, there was no change observed in the levels of Ro mRNAs or La exon 1 mRNA, but a gradual decrease was noted in the mRNA levels of La exon 1'. The importance of alterations in the ratio of La exon 1 to exon 1' is supported by the observations in patients with Sjögren's syndrome, and our results strengthen the notion that the Ro and La antigens participate in the pathogenesis of different autoimmune diseases.

Diagnostic value of the detection of t(14;18) chromosome translocation in malignant hematological and immunopathological diseases using polymerase chain reaction.

The majority of the t(14;18) chromosome translocations that occur in non-Hodgkin centroblastic-centrocytic follicular lymphoma can be detected by various methods. During the translocation process the bcl-2 gene located on chromosome 18 (18q21) is translocated to the JH region of the immunoglobulin gene of chromosome 14 (14q32). The most frequent type of bcl-2 translocations is the mbr type, whereas the immunoglobulin gene breaks mainly at the JH1-6 exons. About one of the 10(5) cells bearing the translocation can already be detected by using nested polymerase chain reaction (PCR). Eight patients suffering from follicular lymphoma were included in this study, which considered the usefulness of the PCR method. The results are in good agreement with those obtained by conventional diagnostic methods. Translocation can be detected, however, in patients with non-malignant diseases such as Sjögren's syndrome (about 5% of the patients) and in a patient with Whipple disease. In addition, translocation was detected in lymphocytes of peripheral blood of a healthy donor. Since lymphomas are detected in patients with Sjögren's syndrome with a relative high frequency, an early diagnosis of the translocation could improve the treatment of the disease. Nevertheless, a diagnosis of lymphoma is valid only in cases of bone marrow translocation-positivity.

Effects of ionic strength on the activity of superoxide dismutase in vitro.

Changes in superoxide dismutase (SOD) activity were studied in vitro at increasing NaCl or KCl concentrations. SOD activity was measured using two different systems of superoxide radical generation: pyrogallol autoxidation, and xanthine-xanthine oxidase reaction. Pyrogallol autoxidation was directly measured by spectrophotometry, whereas in the second case cytochrome c reduction was followed at 550 nm. The inhibition of SOD on those parameters was taken as measure of SOD activity. Increasing concentrations of NaCl and KCI significantly increased the rate of pyrogallol autoxidation. The inhibitory effect of SOD significantly decreased under the influence of these salts and followed an exponential curve. The two salts studied resulted in essentially identical changes in SOD activity. Increasing concentrations of NaCl and KCl decreased the rate of cytochrome c reduction in the xanthine-xanthine oxidase system. When correcting the results for these primary effects, SOD activity also displayed in this system an exponential decay with increasing salt concentrations. The results are interpreted in terms of the known charge distribution pattern on the surface of the SOD molecule, and of the age-dependent increase of the intracellular potassium and sodium concentrations in the postmitotic cells.

In vivo studies on the age-dependent decrease of the rates of total and mRNA synthesis in the brain cortex of rats.

The membrane hypothesis of aging (Zs.-Nagy, I., 1978, J. Theor. Biol. 75, 189-195) attributes the primary role in cellular aging to an age-dependent decrease of the passive potassium permeability of the cell membrane which is due most probably to free-radical damage of the membrane components. As a consequence, the intracellular and intranuclear ionic strength increases resulting in a condensation of the chromatin and a slowing down of the synthetic processes performed by the nucleus. In this concept it was of importance to reveal whether the rates of total and mRNA synthesis display any age-dependent alteration parallel with the change of membrane permeability of the brain nerve cells. Experiments were performed using tritiated uridine incorporation measurements and suitable preparation techniques in young, adult and old rats (1.5, 13 and 25 mth of age, respectively). Comparisons of the incorporation rates revealed a very considerable decrease in the rate of synthesis of both the total and polyadenylated RNA (polyA + RNA) between the ages of 13 and 26 mth. The old animals displayed only about 55 and 67% of the rate of synthesis for the 2 classes of RNA, respectively, as compared to the young and adult rats, if the results are expressed as dpm/mg RNA. However, the decreases are even more pronounced (34 and 41%) if the results are expressed on a dry weight basis. The results obtained are compatible with the membrane hypothesis of aging.

Effect of age on the activity of ceruloplasmin of human blood.

Aminoxidase activity of ceruloplasmin was measured in the serum of 120 people (between 45 and 102 years of age) using the p-phenylenediamine method. A negative linear age-correlation (-30% for the whole lifespan; P < 0.01) was established in this activity with increasing age, nevertheless the total copper content of blood did not change in the same age-range as measured by an atomic absorption spectrophotometric method. Increasing ionic strength, in vitro, caused an exponential decline in blood ceruloplasmin aminoxidase activity of both middle aged and elderly subjects. The age-dependent decrease in ceruloplasmin activity could have a negative effect on the antioxidant functions of blood, and finally on the aging process itself.

In vivo stimulation of nerve cells by phytohemagglutinin. II. Alterations in the rate of total and mRNA synthesis in the brain cortex of old rats.

Bacto-phytohemagglutinin-P (PHA-P) was administered in form of a single intralumbar injection of 2 mg/100 g body weight dose to 24- to 28-mth-old female CGY rats. The accuracy of the injection technique was checked by adding 2% lidocaine to the injection mixture, which resulted in a transient and symmetric paralysis of the posterior limbs when reaching the cerebrospinal fluid. The total RNA content of the liver and brain cortex were measured, and phenolic extraction of RNA was performed the brain cortex. Poly(A) +RNA (mRNA) was separated from the total RNA of the brain cortex by oligo(dT)-cellulose chromatography. Pulse labeling with tritiated uridine was performed 45 min before killing the animals and the incorporation of the radiolabel was measured in the respective RNA classes and corrected for the nucleotide pool size. The rates of total and mRNA synthesis are expressed in percentages of the young untreated rats and compared to old untreated animals. The effects of PHA-P was studied at 4, 10, 20 and 44 h after its injection. A considerable increase of the total RNA content of the brain cortex was measured during the first 10 h of the experiment followed by a slow decrease. However, the RNA content of the brain cortex was still significantly higher at the end of experiment compared to untreated old rats. The rate of total RNA synthesis increased significantly during the first 10 h and remained constantly high until 44 h. The rate of mRNA synthesis increased to a higher extent than that of the total RNA, and also remained high until 44 h.

In vitro studies on the OH* and O2(-*) free radical scavenger properties of idebenone in chemical systems.

OH(*) free radicals were generated by Fenton reaction in the presence of bovine serum albumin (BSA). The decreasing water-solubility of BSA with increasing Fe(2+) concentrations of the system is a sensitive indicator of the cross-linking effects of the OH(*) free radicals. Idebenone (oxidized form) was solubilized for this experiment in DMSO and added to the system in final concentrations of 0.01 or 0.1%. Neither of these concentrations displayed any protective effect against the insolubilization of BSA. Therefore, oxidized idebenone has to be considered as a substance which reacts with OH(*) free radicals slower than the BSA itself, i.e., its oxidized form is not an efficient scavenger of this type of free radicals under the given circumstances. The ability of idebenone to scavenge superoxide radicals was tested in ( [Formula: see text] ) the pyrogallol system; and (ii) the xanthine-xanthine oxidase-nitro blue tetrazolium (XXO-NBT) system. Idebenone did not show any O(2)(-*) radical scavenging ability as revealed by these two in vitro methods, in the concentration ranges studied (up to 75 or 220 microg/ml, respectively). On the contrary, an increasing O(2)(-*) radical generation was observed with increasing concentrations of the drug in both test systems used. The possible biological significance of these observations is discussed in the light of other results like ESR spin trapping and measurements of superoxide dismutase (SOD) activity in various tissues.

The effects of idebenone on DNA and RNA contents as well as synthesis rates of total and poly(A)+ RNA in brain of normal, old C57BL/6J mice and in experimental partial cerebral ischemia of rats.

Effects of idebenone on RNA and DNA contents as well as on synthesis rates of total and poly(A)(+) RNA in the brain were measured in two animal models: (1) Normal young and old, male C57BL/6J mice (6 and 32 months). Idebenone suspended in 5% gum arabic was applied in 50 mg/kg/day dose to old mice for 1 month through a gastric tube. (2) Adult female CFY rats (14-18 months) in which experimental partial cerebral ischemia was induced by bilateral common carotid artery occlusion. Idebenone was administered intraperitoneally in two dose (10mg/kg and 100 mg/kg body weight) 30 min before the interruption of carotid blood flow. DNA content remained invariate during aging in the brain; idebenone treatment did not exert any influence on this parameter. RNA content as well as total and poly(A)(+) RNA synthesis rates, which were measured by incorporation of tritiated uridine into RNA, decreased significantly with age in brain. Idebenone treatment did not cause any essential change of the metabolism of RNA under the given conditions. The RNA and DNA contents of brain were influenced neither by experimental partial cerebral ischemia nor by treatment with idebenone during the ischemia. Partial cerebral ischemia decreased the rate of total and poly(A)(+) RNA synthesis in the brain about 15-45% depending on the methods and basis of expression. This decline could totally be prevented by intraperitoneal application of 10 mg/kg idebenone 30 min before the onset of the partial ischemia. The dose of 100 mg/kg idebenone also elevated the rate of RNA synthesis; however, this increase remained statistically insignificant.

An in vitro model of aging: the influence of increasing physical density on enzyme activities of trypsin, xanthine oxidase and superoxide dismutase.

The enzyme activities of trypsin (using an artificial substrate, Nalpha-benzoyl-L-arginine-ethylester = BAEE), xanthine oxidase (XOD) and superoxide dismutase (SOD) were measured in the absence and presence of various concentrations of the following inert, water-soluble polymer viscogens: polyvinylpyrrolidone (PVP-40), polyethyleneglycol (PEG-6000) and bovine serum albumin (BSA). Enzyme activities measured in the absence of viscogens were taken as 100%. In the presence of the viscogens, enzyme activities decreased considerably as follows: (i) Trypsin: to 2 or 12% in reaction mixtures containing 64 mg/ml PVP-40 or 481 mg/ml PEG-6000, respectively. (ii) XOD: to 29.3% in a reaction mixture containing 116 mg/ml PVP-40, to 68.9% in a medium containing 266 mg/ml PEG-6000, and 38.1% in the presence of 138 mg/ml BSA. (iii) SOD: to 40.0, 19.9 and 16.6% in the same media as listed for XOD, respectively. The observations are consistent with the predictions of the molecular enzyme kinetic model (MEKM), and are also of importance for the membrane hypothesis of aging, since the latter explains the loss of cell functions by an age-dependent increase of intracellular density which may cause serious enzyme inhibitions.

[Detection of t(14;18) chromosome translocation in follicular lymphoma by polymerase chain reaction]. / A t(14;18) kromoszóma transzlokáció kimutatása follicularis lymphomában polimeráz láncreakció segítségével.

In most cases of centroblastic/centrocytic follicular lymphomas the bcl-2 proto-oncogene (18q21) is translocated to the immunoglobulin JH region of chromosome 14 (14q32). About three quarters of the translocations are concentrated on the 3' nontranslated, a few hundred basepare-long region of bcl-2, the so called major breakpoint region (mbr), the remaining 20-25% is located about 30 kilobases downstream of bcl-2 coding sequences in the minor cluster region (mcr). The majority of the immunoglobulin breakpoints can be found in JH6-4 genes. The polymerase chain reaction method can detect the translocation already in a very few number of cells (> 10(3)). This very sensitive technique makes it possible to detect the translocation in lymphoid/lymphoma of peripheral blood and bone marrow that are missed by other diagnostic methods. This way one can perform a quick, early diagnosis, examine the result of treatments as well as detect the remissions and the possible relapses right at the beginning. All the advantages of this method contribute to a more successful treatment of follicular lymphoma. This present work describes a polymerase chain reaction technique which is capable of a detection of the t(14;18) translocation in a patient of centroblastic/centrocytic lymphoma, moreover shows how this translocation disappears after 4 week of radiotherapy of the patient.

[The presence of t(14;18) chromosome translocation in various types of diseases]. / A t(14;18) kromoszóma-transzlokáció jelenléte különbözö típusú betegségekben.

Chromosome translocation of t(14;18) can be detected in most cases of centroblastic/centrocytic follicular lymphomas. They are causative factors of lymphomas but the translocation is present in different other types of diseases although the translocation does not belong to the features of these illnesses. Our present work shows the appearance of t(14;18) translocation in lymphocytes of two patients of Sjögren's syndrome, one that of Whipple disease as well as one of healthy donors' lymphocytes using polymerase chain reaction technique presented in one of our previous publication. The translocation occurred in the mbr of bcl-2 gene in all cases showed and the bcl-2 gene was coupled with the immunoglobulin heavy chain gene. These results are definitively positive concerning the fact of translocation as it has been proved by sequencing of the amplification products showed in our earlier and present paper. Because relatively high percentages of Sjögren's syndrome patients develop later on lymphoma, the early detection of the translocation could result in a more successful diagnosis as well as treatment of the disease. The question arises, however, what role the translocation plays in illnesses such as the Whipple disease or what kind of consequences can be drawn from the appearance of the t(14;18) translocation in lymphocytes of healthy donors.