Synthesis of two DNA-dependent RNA polymerases in yeast

Specific activities of Saccharomyces cerevisiae RNA polymerases I and II were measured in cells growing under different nutrient conditions and throughout the mitotic cell cycle. The specific activity of RNA polymerase I (possibly the ribosomal polymerase) does not vary during the yeast cell cycle. In contrast the specific activity of RNA polymerase II (messenger polymerase) increases during the first third of the cycle and thereafter declines. The independent regulation of synthesis of these two enzymes is further emphasised by observations on the response to different nutrient conditions. Shifting cells from minimal to rich medium led to enhanced RNA polymerase I activity but very little change in activity of RNA polymerase II. Furthermore the activity of RNA polymerase I varies directly with change in growth rate whereas the activity of RNA polymerase II is approximately constant over a range of growth rates. From this data it is suggested: (i) The synthesis of these two enzymes is independently regulated; (ii) RNA polymerase I is synthesised continuously throughout the cycle whereas RNA polymerase II is synthesised periodically early in the cell cycle.     

RNA polymerase activity in isolated nuclei ofNicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg²⁺ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH₄)₂ SO₄ and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.     

BIOLOGICAL ACTIVITY OF THE β NERVE GROWTH FACTOR: THE EFFECTS OF VARIOUS ADDED PROTEINS

—The biological activity of β nerve growth factor varies greatly when other proteins are added to the culture medium. We have found that the biological activity can vary by a factor of 7 in a bioassay using 9 day embryonic chick sensory ganglia by adding bovine serum albumin to the culture medium. Adding the α and γ subunits in the form of 7S nerve growth factor has no effect on the biological activity of the β nerve growth factor. On the other hand, cytochrome c has an intermediate effect. Most of the increase in the biological activity seen on the addition of other proteins seems to be due to the ability of the other proteins to keep β nerve growth factor in solution; i.e. to prevent its adsorption to the culture tubes. However, some of the increase may also be due to an interaction that causes an activation of the β nerve growth factor.     

INFLUENCE OF AGE IN THE INCORPORATION OF [14C]GLYCINE INTO ISOLATED RAT NERVE SEGMENTS IN VITRO

—(1) Sciatic nerves of young rats have been shown to incorporate [¹⁴C]glycine in vitro into the protein fraction at a higher rate than nerves from adult rats under the same conditions. (2) Whilst there is little change in the DNA content of the sciatic nerve of rats with age, there is a is noticeable decline in the RNA content with age. (3) There a linear relationship between the specific activity of the protein fraction and the RNA content of the sciatic nerve under in vitro conditions. (4) There is a decline in the specific activity of the protein fraction with age when expressed against DNA. (5) A linear relationship exists between the logarithm of the specific activity and the length of the femur.     

EVIDENCE OF SYNCHRONIZATION OF NEURONAL ACTIVITY OF MOLLUSCAN BRAIN GANGLIA INDUCED BY ALTERNATING 50 Hz APPLIED MAGNETIC FIELD

Experimental evidence for the appearance of synchronized bioelectric activity in neurons under applied extremely low frequency (ELF) magnetic fields is shown. We have studied the synchronizing process by recording the intracellular bioelectric activity from pairs of neurons randomly chosen from the brain ganglia of the snail Helix aspersa. The recordings were made in real time under exposure to sinusoidal low frequency (50 Hz) weak (B₀=1–15 mT) magnetic fields. Synchronization was observed in 27% of the pairs tested. A linear dependence of the firing frequency f with the energy density of the applied magnetic field (i.e., f∝B₀²) was presented. The ability of low frequency sinusoidal weak magnetic fields to promote “magnetic synchronization” is exciting and opens new avenues for induced electromagnetic field bioeffects.     

Mutually Independent Cyclic AMP and Sodium Responses to Nerve Growth Factor in Embryonic Chick Dorsal Root Ganglia

Abstract: Chick embryo dorsal root ganglia display a rapid and transient rise in their cyclic AMP content when presented with nerve growth factor. These ganglia also depend on nerve growth factor for control of their intracellular Na⁺ and K⁺ levels. A sequential relationship between the cyclic AMP and Na⁺ responses is not readily apparent. Incubation of chick sensory ganglia in a sodium-free medium does not prevent the cyclic AMP response to nerve growth factor from occurring. When ganglia are first incubated with ouabain for 6 h, presentation of nerve growth factor elicits a cyclic AMP response, but no Na⁺ response. The cyclic AMP response therefore does not depend on the Na⁺ environment. An initial presentation of nerve growth factor to the ganglia for 30 min, followed by its withdrawal and subsequent re-administration at different intervals over several hours failed to result in a second cyclic AMP response. Nevertheless, the expected Na⁺ behaviors were still observed. Dibutyryl cyclic AMP is capable of eliciting a cyclic AMP response in chick sensory ganglia after 6 h of nerve growth factor deprivation. When both agents were presented simultaneously to the ganglia, only a single cyclic AMP response was obtained, corresponding in time to the response elicited by dibutyryl cyclic AMP alone-indicating that this drug acts on the NGF-sensitive cells. At the same time dibutyryl cyclic AMP alone failed to result in a Na⁺ response, leading one to conclude that the cyclic AMP response to nerve growth factor is truly not mediating the Na⁺ response. Additional support for the mutual independence of these two short-latency responses is provided by the apparent inability of nerve growth factor to cause a cyclic AMP response in chick embryo sympathetic ganglia, another traditional target for the factor, which is capable of displaying a Na⁺ response.     

DIVALENT CATION SPECIFICITY OF THE CALCIUM REQUIREMENT FOR FAST TRANSPORT OF PROTEINS IN AXONS OF DESHEATHED NERVES

The presence of a requirement for calcium during the fast transport of [³H]protein in axons was assessed in desheathed spinal nerves of bullfrog. The nerves were desheathed locally along 4 mm of their length, and desheathing was judged effective on the basis of an enhanced uptake of [³H]leucine into that region of nerve trunk. Desheathing per se had a slight inhibitory effect on transport. Incubation of desheathed nerve trunks in calcium-free medium reduced transport by 60-80% relative to that in desheathed nerves incubated in normal medium. Addition of Mg²⁺ or Sr²⁺ to the calcium-free medium allowed transport to proceed normally. Addition of Co²⁺ or Mn²⁺ to normal medium did not affect transport in desheathed isolated nerve trunks. When ganglia and nerve trunks were both incubated in medium containing 0.18 mM-CoCl₂, transport was depressed to a similar extent proximal and distal to the desheathed region. This again indicates that Co²⁺ does not inhibit transport in desheathed nerves, whereas it does inhibit transport in the ganglia. Additive inhibitory effects were observed when ganglia were incubated in medium containing 0.018 mM-CoCl₂, and desheathed nerves were incubated in calcium-free medium. Differences in the divalent cation specificities of the axonal and ganglionic calcium requirements suggest that calcium supports transport in nerves in a manner distinct from its role in maintaining transport in spinal ganglia. It is concluded that the ganglionic calcium requirement involves initiation of axonal transport in the soma rather than translocation in the intraganglionic region of axon.     

PATTERN OF RNA SYNTHESIS IN THE SCIATIC NERVE OF THE HEN DURING WALLERIAN DEGENERATION

The pattern of synthesis of rapidly-labelled RNA of hen sciatic nerve was studied during Wallerian degeneration. At 2,4,8, 16 and 30 days of degeneration the proximal and distal stumps of the severed nerve as well as the intact contralateral sciatic nerve (functional control) were excised and incubated with either [5-³H]uridine or [2-¹⁴C]uridine for 0.5 h. The electrophoretic pattern of RNA from the normal adult sciatic nerve showed that most of the radioactivity was incorporated into RNA species migrating between the 18 S and 4 S components of the bulk RNA. The synthesis of RNA was sensitive to actinomycin-D, an indication that it was directed by a DNA template. The electrophoretic patterns of the rapidly-labelled RNA in the proximal and distal nerve stumps demonstrated a change following nerve section. After 2–4 days of Wallerian degeneration the degenerating distal nerves incorporated more radioactivity in the 4 S region than the corresponding controls, but at 8 and 16-days after degeneration relatively more label appeared in higher molecular weight RNA species. In the intact sciatic nerve of the operated hens progressively more radioactivity was detected in the 4 S region with increasing time after the contralateral nerve section. At each stage of Wallerian degeneration the specific radioactivities of RNA in the control nerves from experimental hens were higher than those of the normal adult sciatic nerve. These results indicated a change of RNA metabolism in increased functional activity and during Wallerian degeneration.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

VARIABILITY IN RATIOS OF PHYTOPLANKTON CARBON AND RNA TO ATP AND CHLOROPHYLL A IN BATCH AND CONTINUOUS CULTURES1,2

The feasibility of estimating phytoplankton carbon and RNA concentrations from measurements of ATP and chlorophyll a (chl a) concentrations was studied using chemostat populations of the marine diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle (= T. fluviatilis Hustedt). C:ATP and RNA:ATP ratios were studied for six additional marine species in batch culture representing five classes of phytoplankton. Statistical analyses revealed that both the growth rate and the factor limiting growth (NO₃^-, NH₄⁺, PO₄^3- or light) could alter C:ATP, RNA: ATP, C:chl a and RNA:chl a ratios by amounts which were large compared to measurement error. An analysis of variance of the batch culture results indicated that both species and the source of inorganic nitrogen (NO₃^-, or NH₄⁺) had a significant effect on C:ATP and RNA:ATP ratios. Light had less of an influence on C:ATP and RNA:ATP ratios than on C:chl a and RNA:chl a ratios, and for this reason we feel that phytoplankton C and RNA concentrations can be estimated with greater reliability from ATP than from chl a measurements. The range of C:ATP and RNA:ATP values found for T. weissflogii under a variety of growth conditions was similar to that for the six additional species grown in batch culture, suggesting that this range of values is indicative of the extremes likely to occur in living cells. Our results and additional data in the literature indicate that phytoplankton C and RNA concentrations can be estimated to within a factor of two by multiplying ATP concentrations by 311 and 35, respectively, in N limited systems, and by 341 and 36, respectively in PO₄^3- limited systems.     

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE,GONYAULACALES)1

The low CO₂ concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO₂ availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO₂ concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO₂]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO₃^? under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO₂]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO₂]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO₂ accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO₂] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO₂.     

EFFECT OF AN INHIBITING FACTOR ISOLATED FROM RAT LIVER ON DNA POLYMERASES IN REGENERATING RAT LIVER

We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G₁—S transition, and reduced in vivo [³H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented α DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on α DNA polymerase was detected. LIF did not affect β DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Selective Modulation of RNA Polymerase I Activity during Growth Transitions in the Soybean Seedling

RNA polymerase I and II activities were measured in tissues of the soybean (Glycina max, var. Wayne) hypocotyl where dramatic changes in the relative level of RNA synthesis are associated with normal and auxin-induced growth transitions. When assayed in isolated nuclei, the activity of RNA polymerase I changed much more than the activity of RNA polymerase II during these growth transitions. The activity of RNA polymerase I expressed in the nuclei generally showed a positive correlation with the relative level of RNA synthesis (i.e. accumulation) of that tissue. Following solubilization of the RNA polymerases from these isolated nuclei and fractionation of them on DEAE-cellulose, the activity of RNA polymerase I relative to that of RNA polymerase II showed smaller changes during these growth transitions than when assayed in the nuclei. Thus, these data indicate that the activity of RNA polymerase I is significantly modulated in the nucleus, up or down depending upon the growth state, during growth transitions in the soybean in addition to lesser changes which occur in the apparent level of the enzyme.     

RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg²⁺ with optima at ? 0.01 and 0.02 M MgCl₂, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl₂ concentrations. In the presence of 0.01 M MgCl₂ the optimum (NH₄)₂SO₄ concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [₃H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated ? 3 pmoles of [₃H]UMP/muml; DNA at 25 C for 15 min, and ? 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, α-amanitin (20 m?/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with α-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).     

Immunocytochemical characterization of human NOR-90 (upstream binding factor) and associated antigens reactive with autoimmune sera

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to the human upstream binding factor (hUBF) and composed of twoMr forms. In this study, thirteen human anti-NOR-90/hUBF autoimmune sera were used to further characterize NOR-90/hUBF and its associated autoantigens. Nucleolar and nucleoplasmic staining of interphase cells and NOR staining in mitosis were observed with all sera by immunofluorescence. All sera showed equal reactivity with both high and lowMr forms in Western blotting and immunoprecipitation, suggesting that the cellular content and distribution for bothMr forms were approximately equal. Using extracts of [³⁵S]methionine- and [³²P]orthophosphate-labeled cells, phosphorylated and nonphosphorylated NOR-90/hUBF were identified for bothMr forms and these two populations were recognized by human autoantibodies. In immunoprecipitation analyses, the nonphosphorylated population was readily extracted while the phosphorylated population was tightly bound. Clinical data were available for 8 patients in whom anti-NOR-90/hUBF autoantibodies were present. They had diverse diagnoses including SLE, rheumatoid arthritis and malignancies. Although only one patient was diagnosed as scleroderma, Raynaud's phenomenon was observed in 4 of the 8 patients. Interestingly, one NOR-90/hUBF serum was shown to contain additional antibodies to RNA polymerases I and II.Abbreviations: ANA=Antinuclear antibody; HCC=hepatocellular carcinoma; hUBF=human upstream binding factor; NOR=nucleolus organizer region; RNA pol I=RNA polymerase I; RNA pol II=RNA polymerase II; rRNA=ribosomal RNA; SLE=systemic lupus erythematosus.Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto-Shi 390, Japan