[Insomnia--state of the science]. / Insomnien--Stand der Forschung.

Diagnostic systems such as the international classification of diseases (ICD-10) or the diagnostic and statistical manual of mental disorders (DSM IV) have frequently been criticized as not adequately reflecting the complexity and heterogeneity of insomnia. Progress was made through the introduction of the international classification of sleep disorders (ICSD-2) and the research diagnostic criteria (RDC). The DSM-5 introduced the new category of insomnia disorder, thus relinquishing the traditional dichotomy of primary versus secondary insomnia. Recent basic research indicates that genetic and epigenetic factors are involved in the etiology of insomnia; the so-called three P model (i.e. predisposing, precipitating and perpetuating factors) and the hyperarousal concept have gained much attention in trying to explain the pathophysiology of insomnia. With respect to the cognitive-behavioral therapy of insomnia (CBT-I), a plethora of empirical evidence supports the first-line character of this type of treatment for insomnia. Unfortunately, CBT-I is still administered to only a minority of afflicted patients, probably due to a lack of resources in the healthcare system. As a consequence, stepped-care models to improve insomnia therapy encompass self-help programs, internet-based treatment avenues, community-centered activities (specially trained nurses) and as a last resort medical specialists/psychotherapists and sleep experts to deal with insomnia.

REM sleep instability--a new pathway for insomnia?

Chronic insomnia afflicts approximately 10% of the adult population and is associated with daytime impairments and an elevated risk for developing somatic and mental disorders. Current pathophysiological models propose a persistent hyperarousal on the cognitive, emotional and physiological levels. However, the marked discrepancy between minor objective alterations in standard parameters of sleep continuity and the profound subjective impairment in patients with insomnia is unresolved. We propose that "instability" of REM sleep contributes to the experience of disrupted and non-restorative sleep and to the explanation of this discrepancy. This concept is based on evidence showing increased micro- and macro-arousals during REM sleep in insomnia patients. As REM sleep represents the most highly aroused brain state during sleep it seems particularly prone to fragmentation in individuals with persistent hyperarousal. The continuity hypothesis of dream production suggests that pre-sleep concerns of patients with insomnia, i. e., worries about poor sleep and its consequences, dominate their dream content. Enhanced arousal during REM sleep may render these wake-like cognitions more accessible to conscious perception, memory storage and morning recall, resulting in the experience of disrupted and non-restorative sleep. Furthermore, chronic fragmentation of REM sleep might lead to dysfunction in a ventral emotional neural network, including limbic and paralimbic areas that are specifically activated during REM sleep. This dysfunction, along with attenuated functioning in a dorsal executive neural network, including frontal and prefrontal areas, might contribute to emotional and cognitive alterations and an elevated risk of developing depression.

Mindfulness and self-compassion in dermatological conditions: a systematic narrative review.

OBJECTIVE: People affected by chronic skin conditions suffer from elevated levels of psychological distress. There is a need for evidence-based treatments that integrate medical care. Mindfulness and Self-compassion programs (MCBPs) have proven effective in chronic diseases. This systematic review aims to narratively synthesize the literature on mindfulness and self-compassion as traits and interventions in chronic skin conditions. DESIGN: We searched four electronic databases for mindfulness and self-compassion trials and correlational studies in chronic skin conditions. We narratively synthetized results regarding the effects of mindfulness and self-compassion, both as traits and as interventions, on psychological and disease outcomes. RESULTS: Thirteen studies were included in our review. Evidence from cross-sectional studies suggest that mindfulness and self-compassion are linked to lower psychological distress and better adjustment to the disease. MCBPs appear feasible for this population and can lower psychological distress, reduce disease severity and improve quality of life. Methodological issues limit conclusions on MCBP efficacy. Based on our analysis, we propose possible mechanisms that future research could explore. CONCLUSIONS: The integration of MCBPs in the care process of chronic skin conditions appears promising. Definitive conclusions cannot be drawn due to a lack of strong evidence. Further studies with high methodological standards are needed.

[Lack of sleep and insomnia. Impact on somatic and mental health]. / Schlafmangel und Insomnie. Einfluss auf die körperliche und psychische Gesundheit.

Lack of sleep and insomnia need to be viewed differently. Lack of sleep implies a shortening of the habitual sleep duration due to external circumstances or motivational factors. Insomnia, in contrast, is defined as a sleep disorder due to unknown reasons for the afflicted subjects. People with insomnia suffer from being unable to sleep, in spite of adequate external circumstances. Research on lack of sleep/shortened sleep duration has focused on relationships with somatic and mental health. Longitudinal studies revealed that a shortening of sleep duration (< 6 h) is associated with an increased risk for the metabolic syndrome and cardiovascular diseases. For sleep duration and mortality, a U-shaped relationship was found, indicating that both shortened (< 6 h) and prolonged sleep durations (> 8 h) are associated with increased mortality. Similar, albeit weaker, correlations were described for insomnia and somatic health. In addition, insomnia is a risk factor for the development of mental disorders, especially depression. These relationships suggest that the area of sleep and sleep disorders should be integrated into everyday medical practice and that preventive approaches to somatic and mental disorders should encompass the topic of sleep to a much stronger extent than currently practiced.

A study of immunoglobulin structure. II. The comparison of Bence Jones proteins by peptide mapping.

Urinary proteins of patients with myeloma, prepared by precipitation with ammonium sulphate, have been separated by gel filtration on Sephadex G-100 after reduction and aminoethylation. Many specimens separated into a major peak of Bence Jones protein and into minor peaks of albumin, a protein tentatively identified with heavy chain and a smaller molecular weight protein corresponding to the variable portion of the corresponding Bence Jones protein. The Bence Jones protein purified by gel filtration was analyzed by electrophoresis and by peptide mapping after tryptic digestion. The peptide maps of 24 type K and 20 type L Bence Jones proteins were compared. A set of common peptides was identified in the peptide maps of the Bence Jones proteins of the same type; the common peptides of type K proteins were completely different from the common peptides of type L proteins. The patterns of distinctive peptides was compared; no similarities were found between distinctive peptides of type K and of type L proteins. Some similarities were observed in the distinctive peptides of proteins of the same type. The similarities involved in many cases peptides containing cysteine, whereas similarities in other peptides were limited. This observation suggested that the amino acid sequence around the cysteines of the variable NH(2)-terminal half of the Bence Jones proteins may show less variability than other sequences. A few proteins of the same type differed in all their distinctive peptides, an indication that multiple amino acid differences exist between individual Bence Jones proteins. The genetic mechanisms responsible for the variability in the amino acid sequence of the NH(2)-terminal half of the light chains of immunoglobulins are discussed in view of the results of the comparison by peptide mapping of the Bence Jones proteins.

Catabolic origin of a Bence Jones protein fragment.

Gel filtration analysis of the urinary proteins of some patients with myeloma has shown the presence of "fragments" of Bence Jones proteins which correspond to the variable half of these proteins. Experiments have been carried out to establish the origin of a "fragment" observed in a patient who excreted a large amount of this protein. Labeled homologous Bence Jones protein has been injected into this and other control patients. Excretion of labeled "fragment" has been observed in all. Analysis by peptide mapping and radio-autography of this labeled "fragment" isolated from the urine showed that the invariable half of the Bence Jones protein was not excreted; it seemed thus likely that the invariable half was metabolized to small peptides and free amino acids. A labeled Bence Jones protein which was excreted without any accompanying "fragment" was injected into the patient who excreted large amounts of "fragment." No excretion of labeled "fragment" was observed. It was thus concluded that the property of being degraded to "fragment" is characteristic of some "fragile" Bence Jones proteins and is not determined by the patient. Incubation with serum or urine of the "fragile" Bence Jones protein failed to produce any "fragment." "Fragments" of Bence Jones proteins are thus most likely formed during excretion of these proteins through the kidney and are products of the catabolism of Bence Jones proteins.

The AU-rich sequences in the 3' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids.

Different vectors were constructed that expressed the human interferon-beta (IFN-beta) mRNA constitutively and contained various deletions in the 3' untranslated region (UTR). AU-rich sequences in the 3' UTR were specifically deleted in two vectors. Cell lines secreting human IFN-beta were established by transfecting murine L929 cells with the vectors. These cells showed similar levels of IFN-beta mRNA and secreted comparable amounts of IFN-beta, indicating that the deletion of AU-rich sequences had no effect on the stability and little effect on the efficiency of translation of this mRNA. The synthetic glucocorticoid dexamethasone was previously shown to increase the turnover of IFN-beta mRNA. This activity of dexamethasone was clearly observed only in cells expressing IFN-beta mRNA with AU-rich sequences in the 3' UTR. The increased turnover of this mRNA occurred in the presence of cycloheximide; therefore, it did not require synthesis of new proteins. These findings suggest that glucocorticoids may activate a ribonuclease that degrades mRNAs containing AU-rich sequences in the 3' UTR.

A study of immunoglobulin structure. 3. An estimate of the variability of human light chains.

102 human Bence Jones proteins have been purified by gel filtration, digested with trypsin, and analyzed by peptide mapping. In several cases Bence Jones "fragments", corresponding to the variable half of the corresponding proteins, were observed. The peptide maps of the proteins were compared to establish whether any identical proteins were present in the sample analyzed. No Bence Jones protein showed a peptide map identical to that of any other protein, although remarkable similarities in the peptide maps were observed for some proteins. Two proteins that gave very similar peptide maps were then examined in detail, by purifying and analyzing the tryptic peptides. It was then found that these two proteins differ in amino acid sequence in at least six positions. The probability of not finding two identical sequences by examining a sample extracted from populations of light chains of different sizes has been calculated. This has led to an estimate of the minimal size of the population of light chain sequences in humans. The number of light chain sequences appears to be at least a few thousand. Information on the frequency of Inv and Oz antigenic determinants and on the relative frequency of subtypes of K chains has been obtained. Proteins of KI subtype are found most frequently. The possibility that different subtypes may be predominant in different species is discussed in relation to the evolutionary arguments used in favor of the somatic theories on the origin of variability of immunoglobulin chains.

Synthesis and release of platelet-activating factor is inhibited by plasma alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin and is stimulated by proteinases.

TNF and IL-1 stimulate the synthesis and release of platelet-activating factor (PAF) by neutrophils and vascular endothelial cells. Serum inhibits PAF production even after inactivation of an acetylhydrolase that degrades PAF. Human plasma was fractionated by gel filtration chromatography, and two inhibitory fractions were detected, one containing PAF-acetylhydrolase activity and the other alpha 1-proteinase inhibitor. Low concentrations of this antiproteinase and of human plasma alpha 1-antichymotrypsin inhibited TNF-induced PAF synthesis in neutrophils, macrophages, and vascular endothelial cells. Both antiproteinases also inhibited PAF production stimulated by phagocytosis in macrophages and induced with IL-1 in neutrophils or with TNF in vascular endothelial cells. These results suggest that a proteinase activated on the plasma membrane or secreted by these cells is involved in promoting PAF synthesis. Indeed, addition of elastase to macrophages, neutrophils, and endothelial cells stimulated synthesis and release of PAF much faster than TNF. A similar stimulation was observed in incubations with cathepsin G. To identify a proteinase activated in TNF-treated cells, neutrophils and endothelial cells were incubated with specific chloromethyl ketone inhibitors of elastase and cathepsin G. Synthesis of PAF was significantly inhibited by low concentrations of the cathepsin G inhibitor. The finding that antiproteinases are inhibitory at concentrations 100-fold lower than those present in plasma raises questions as to the ability of TNF and IL-1 to stimulate neutrophils in circulation or endothelial cells to synthesize PAF. We propose that PAF production is limited to zones of close contact between cells, which exclude antiproteinases.

Antiinflammatory peptides (antiflammins) inhibit synthesis of platelet-activating factor, neutrophil aggregation and chemotaxis, and intradermal inflammatory reactions.

Synthetic peptides corresponding to the region of highest similarity between human lipocortin I and rabbit uteroglobin inhibit phospholipase A2 and show potent antiinflammatory activity on the carrageenan-induced rat footpad edema. The peptide HDMNKVLDL (antiflammin-2) inhibits the synthesis of platelet-activating factor (PAF) induced by TNF or phagocytosis in rat macrophages and human neutrophils, and by thrombin in vascular endothelial cells. The peptide MQMKKVLDS (antiflammin-1) is less inhibitory than antiflammin-2 for macrophages and not inhibitory for neutrophils after a 5-min preincubation. This finding suggests that antiflammin-1 is inactivated by neutrophils secretory products, possibly oxidizing agents. Synthesis of PAF is inhibited by antiflammin-2 without an appreciable lag, but this inhibition is reversed when neutrophils or macrophages are washed and incubated in fresh medium. Therefore, antiflammins must be continuously present to inhibit PAF synthesis. Antiflammins block activation of the acetyltransferase required for PAF synthesis, suggesting that this enzyme is another target for the inhibitory activity of antiflammins. These peptides inhibit neutrophil aggregation and chemotaxis induced by complement component C5a. Antiflammin-2 suppresses the increase in vascular permeability and the leukocyte infiltration induced in rats by an Arthus reaction or by intradermal injection of rTNF and C5a.

Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor.

Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.

Allelic antigenic factor Inv(a) of the light chains of human immunoglobulins: chemical basis.

Twenty-seven Bence Jones proteins of immunological type K show a common set of peptides. One 6f the commnon peptides differs in three of the proteins which are the only ones classified by a serological test as Iav(a+). The difference in the peptide analyzed is caused by a valine-leucine interchange; Inv(a+) proteins have leucine, whereas Inv(a-) proteins have valine in position 189.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Heterogeneous nuclear RNA promotes synthesis of (2',5')oligoadenylate and is cleaved by the (2',5')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.

Correlation between growth inhibition and presence of 5'-methylthioadenosine in cells treated with interferon.

Treatment with interferon of several lines of human and murine cells resulted in a modest increase of the S- adenyosylmethionine level and in a greater increase of the S-adenosylhomocysteine level, with a consequent decrease in the S- adenyosylmethionine /S-adenosylhomocysteine ratio. These results confirmed a previous report (F. de Ferra and C. Baglioni , J. Biol. Chem., 258: 2118-2121, 1983) of such an effect of interferon on HeLa cells. The increase in S- adenyosylmethionine and S-adenosylhomocysteine was not observed in lymphoblastoid Raji cells, which are not growth inhibited by interferon. When Daudi cells and two other cell lines sensitive to growth inhibition by interferon were labeled with [35S]methionine, two novel labeled compounds were detected by high-pressure liquid chromatography analysis of cell extracts. These compounds were not present in untreated cells, and were either absent or present in reduced amount in extracts of cells resistant to the antiproliferative effect of interferon. One of the labeled compounds was tentatively identified with 5'-methylthioadenosine (MTA), a naturally occurring nucleoside, which was previously shown to inhibit murine lymphoid cell proliferation. Addition of MTA to cultures of different cell lines resulted in inhibition of cell growth. When the MTA concentration was measured in Daudi cells treated in this way, a relatively low concentration of MTA was detected, which was comparable to that found in interferon-treated cells. It seems possible that the presence of MTA in interferon-treated cells contributes to the inhibition of cell growth.

Induction of class I major histocompatibility complex antigens in human teratocarcinoma cells by interferon without induction of differentiation, growth inhibition, or resistance to viral infection.

The behavior of human teratocarcinoma cells, and especially their stem cells (embryonal carcinoma cells), may provide insights into the properties of human early embryonic cells. We report here that human recombinant gamma-interferon (IFN-gamma) induced the expression of major histocompatibility complex Class I (HLA-A, B, C) antigens and beta 2-microglobulin in the two human embryonal carcinoma cell lines, 2102Ep cl.4D3 and NTERA-2 cl.D1, and in the yolk sac carcinoma cell line, 1411H; human recombinant IFN-alpha and IFN-beta were less effective inducers of these cell surface molecules. No induction was observed in the gestational choriocarcinoma cell line, JAR. Neither IFN-alpha, IFN-beta, nor IFN-gamma caused growth inhibition, expression of major histocompatibility complex Class II (HLA-DR) antigens, resistance to viral (vesicular stomatitis virus) infection, or expression of 2',5'-oligo(A)synthetase in any of the cells. Also, IFN-gamma neither induced differentiation of NTERA-2 cl.D1 cells, which are pluripotent human stem cells, nor influenced their differentiation induced by retinoic acid. However, developmental regulation of responsiveness to IFN was evident, since IFN-gamma induced higher levels of surface expression of HLA-A, B, C and beta 2-microglobulin in the retinoic acid-induced differentiated NTERA-2 cl.D1 cells than in the undifferentiated parental cells. Also, 2',5'-oligo(A)synthetase was inducible in the NTERA-2 cl.D1 differentiated cells by IFN-alpha and -beta, although not by IFN-gamma, and slight resistance to vesicular stomatitis virus infection was evident in aged cultures of differentiated cells exposed to IFN-alpha. The effect of recombinant mouse IFN-gamma on major histocompatibility complex expression by several murine teratocarcinoma cells was also examined: H-2 Class I (H-2Db), but not class II (I-Ab), antigens were induced in the parietal yolk sac carcinoma lines, PYS and F9Ac cl.9; in cultures of PCC3/A/1 containing both embryonal carcinoma (EC) and differentiated cells; and in cultures of the EC cells, PCC4azaR and PCC4AO, without evidence of differentiation. No induction was observed in the murine EC cell lines, F9 or FA (H-2Kk). Our results indicate that human EC cells, like murine EC cells, exhibit only a partial response to the interferons, and that the extent of this response is developmentally regulated.

Expression of messenger RNAs for complement inhibitors in human tissues and tumors.

The mRNAs coding for three complement inhibitors produced by human cells, complement cytolysis inhibitor (CLI), decay-accelerating factor (DAF), and CD59, are characteristically distributed among normal tissues. High levels of CLI mRNA are expressed in tissues that express low levels of DAF mRNA and vice versa. Therefore, the expression of these mRNAs shows a mutually exclusive relationship, with the possible exception of the lung, where all these mRNAs are expressed. In contrast, CD59 mRNA is rather uniformly expressed in all tumor cell lines examined, whereas the mRNA for either of the two other complement inhibitors is overexpressed in some specific tumor cells, e.g., HeLa cells overexpress DAF mRNA, while A172 cells overexpress CLI mRNA. These two cell lines were resistant to antibody-dependent complement cytotoxicity. Expression of CLI and DAF mRNA was induced in cells treated with the antitumor drug N-(chloroetyl)-N'-cyclohexyl-N-nitrosourea; these cells became resistant to complement cytotoxicity. A similar pattern of expression was detected in tumor samples obtained during surgery, with a relatively uniform expression of CD59 mRNA and occasional overexpression of CLI or DAF mRNA. These findings suggest that overexpression of complement inhibitors mRNA and of the corresponding proteins may contribute to tumor cell resistance to complement-mediated cytotoxicity.

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.

Differential induction and decay of manganese superoxide dismutase mRNAs.

Human manganese superoxide dismutase (MnSOD) is encoded by two mRNAs of 4 and 1 kb, respectively. These mRNAs are transcribed from the same gene and have an identical coding sequence, but differ in the 3' untranslated sequence because of alternate polyadenylation. Tumor necrosis factor-alpha (TNF) induced both 4- and 1-kb mRNAs in all the human cell lines examined. However, the relative expression of these mRNAs varied significantly among different cell lines after an 8-h treatment with TNF. Therefore, the time course of induction by TNF and the decay of MnSOD mRNAs after TNF removal were analyzed. The rate of accumulation of the 4-kb mRNA was initially much greater than that of the 1-kb mRNA, suggesting that the 4-kb mRNA was produced faster than the 1-kb mRNA. The rapid accumulation of the 4-kb mRNA was offset after a few hours by an enhanced rate of decay. The half-life of the 4-kb mRNA was approximately 2-4 h in different cells while that of the 1-kb mRNA was approximately 10-12 h. This different half-life of mRNAs that encode the same protein suggests that their relative expression is also regulated by a post-transcriptional mechanism affecting their turnover. Additional evidence supporting the differential decay of the two MnSOD mRNAs was obtained by incubation in a rabbit reticulocyte cell-free system; the 4-kb mRNA decayed rapidly while the 1-kb mRNA appeared to be stable.