Effects of switching weekly alendronate or risedronate to monthly minodronate in patients with rheumatoid arthritis: a 12-month prospective study.

UNLABELLED: Switching weekly ALN or RIS to monthly MIN in patients with RA, of whom two-thirds were treated with low-dose PSL, significantly decreased bone turnover markers and increased BMD at 12 months, suggesting that monthly MIN may be an effective alternative treatment option of oral bisphosphonate treatment. INTRODUCTION: The aim of this prospective, observational study was to evaluate the effects of switching weekly alendronate (ALN 35 mg) or risedronate (RIS 17.5 mg) to monthly minodronate (MIN 50 mg) in patients with rheumatoid arthritis (RA). METHODS: Patient characteristics were as follows: n = 172; 155 postmenopausal women, age 65.5 (44­87) years; T-score of lumbar spine (LS), −1.4; total hip (TH), −1.8; femoral neck (FN), −2.1; dose and rate of oral prednisolone (2.3 mg/day), 69.1 %; prior duration of ALN or RIS, 46.6 months; were allocated, based on their preference, to either the (1) continue group (n = 88), (2) switch-from-ALN group (n = 44), or (3) switch-from-RIS group (n = 40). RESULTS: After 12 months, increase in BMD was significantly greater in group 3 compared to group 1: LS (4.1 vs 1.2 %; P < 0.001), TH (1.9 vs −0.7 %; P < 0.01), and FN (2.7 vs −0.5 %; P < 0.05); and in group 2 compared to group 1: LS (3.2 vs 1.2 %; P < 0.05) and TH (1.5 vs −0.7 %; P < 0.01). The decrease in bone turnover markers was significantly greater in group 3 compared to group 1: TRACP-5b (−37.3 vs 2.5 %; P < 0.001), PINP (−24.7 vs −6.2 %; P < 0.05), and ucOC (−39.2 vs 13.0 %; P < 0.05); and in group 2 compared to group 1: TRACP-5b (−12.5 vs 2.5 %; P < 0.05) at 12 months. CONCLUSIONS: Switching weekly ALN or RIS to monthly MIN in patients with RA may be an effective alternative treatment option of oral bisphosphonate treatment.

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells.

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.

Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes.

N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA. The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it. The alkylated RNA base was found to be adenine. The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.

Partial characterization of RNA polymerase II complex released by micrococcal nuclease digestion of rat liver nuclei.

Two forms of RNA polymerase II were released from rat liver chromatin by micrococcal nuclease digestion of the nuclei. One from behaved like a free RNA polymerase II and the other like a complex with other nuclear components. Both forms of RNA polymerase II activity were recovered in the 0.16 M NaCl-soluble fraction of the nuclear digest, and the complexed from the RNA polymerase II could transcribe its endogenous template under conditions permitting only of elongation of the RNA synthesis. The RNA polymerase II complex was further purified by gel filtration chromatography and column electrophoresis. Analysis of protein and DNA of the partially purified complex suggested that the RNA polymerase II was bound to mono- or dinucleosomes carrying some characteristic nonhistone proteins. Furthermore, in experiments on tissues from starved rats, the two forms of RNA polymerase II were found to originate from different functional states of the chromatin-bound enzyme in vivo.

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60.

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.

Increased micrococcal nuclease sensitivity and/or solubility in nuclei from Graves' disease thyroid tissue.

The sensitivity of nuclei to micrococcal nuclease was compared in thyroid tissue obtained from euthyroid patients with solitary cold nodules and from patients with Graves' disease. A significant increase in the solubility and/or the sensitivity of nuclei to the nuclease was found in thyroid tissue from patients with Graves' disease. Electrophoretic analysis of DNA in chromatin solubilized by the nuclease revealed that the amount of oligonucleosomal DNA was increased, and that of polynucleosomal DNA was even more increased, in nuclei from Graves' thyroids than in those from normal thyroids. Polyacrylamide gel electrophoretic analysis in Triton acid-urea showed that the extent of histone acetylation in nuclei from Graves' thyroids was almost the same as in those from normal thyroids. These findings suggest that the state of chromatin organization in Graves' thyroid nuclei is different from that in normal thyroid nuclei and is independent of the extent of histone acetylation.

Heterogeneity of protein kinase NII. Multiple subunit-polypeptides.

Protein kinase NII has a alpha alpha' beta 2 subunit structure, and consists of a chromatographically heterogeneous population. By two-dimensional polyacrylamide gel electrophoresis, each subunit was resolved into multiple polypeptides with various pI values: alpha subunit, 4 spots; alpha' subunit, 10 spots; and beta subunit, 4 spots. NII underwent autophosphorylation on beta subunits. Fractions of alpha and alpha' polypeptides also occurred as phosphoforms as shown by alkaline phosphatase treatment. In addition, alpha' subunit had another motif for heterogeneity, which separated alpha' polypeptides into two groups, and was exemplified by NIIa and NIIb that showed different enzyme kinetics and the nuclear localization. We interpret these to account for the basis of the functional as well as molecular heterogeneities of protein kinase NII.

Transcribing chromatin is not preferentially enriched with acetylated histones.

Chromatin fragments of the RNA polymerase II-transcriptional complex were purified from the micrococcal nuclease digest of rat liver nuclei in the presence of n-butyrate, a potent histone deacetylase inhibitor. Polyacrylamide gel electrophoretic analysis in Triton acid-urea revealed that the extent of histone acetylation of the complex did not differ markedly from that of the total chromatin.

Characterization of a 42 degrees C-specific heat shock protein of mammalian cells.

HeLa cells synthesize a particular heat shock protein that is induced only by heat shock at 42 degrees C, and not at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). We further characterized the 42 degrees C-specific protein. This protein was induced in mouse FM 3A cells as well as in human HeLa cells. In both cell lines, the protein was resolved into two spots, a basic polypeptide and an acidc one. The mRNA of the protein was induced during the incubation of these cells at 42 degrees C, and the in vitro translation product of mRNA corresponded to the basic, not to the acidic, polypeptide. During the chase period for cells that were labeled with [35S]-methionine, the basic polypeptide of the protein decreased, and the acidic one increased, indicating that the protein was synthesized as the basic polypeptide and then somehow modified to become the acidic one. The 42 degrees C-specific protein was found only in the cytosol fraction, and not in the nuclear or other particulate fractions, in both HeLa and FM 3A cells. The results suggested that the 42 degrees C-specific protein may have some function in the cytoplasm of mammalian cells during mild heat shock.

Characteristic distribution of two forms of chromatin-bound RNA polymerase II in rat liver nuclei.

We have shown that, in rat liver nuclei, the chromatin-bound RNA polymerase II is released as two different forms on digestion with micrococcal nuclease or DNase I (peak 1 and peak 2). To elucidate the origin of the two forms of the enzyme, we examined their distribution in fractionated chromatins obtained by mild micrococcal nuclease digestion of the nuclei. About half of the total peak 2 activity was recovered in a nuclease-sensitive chromatin fraction which contained DNA enriched in the sequences appearing in the polysomal polyadenylated mRNA. On the other hand, four-fifths of the total peak 1 activity was recovered in a nuclease-resistant chromatin fraction which contained DNA comprising only two thirds of the transcribed sequences. Furthermore, during the nuclease digestion, peak 2 activity was rapidly released from the chromatin, whereas peak 1 activity was gradually released. These results indicate that the two forms of RNA polymerase II are distributed differently in the cell nuclei.

Induction of four heat-shock proteins and their mRNAs in rat after whole-body hyperthermia.

When the body temperature of rats was brought to 42 degrees C, four heat-shock proteins, with molecular weights of 70,000, 71,000, 85,000, and 100,000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), were induced in various tissues of the rats. The hsp 70 was strongly induced by hyperthermia, and its accumulation was detected by Coomassie blue staining. The hsp 71 was abundant in various tissues of rats that were not heat-shocked. Analysis of translation products of liver mRNAs from heat-shocked rats also showed increased synthesis of the four heat-shock proteins, indicating that these hsp-mRNAs were induced after hyperthermia. Induction of the hsp-mRNAs was transient after hyperthermia. The four heat-shock proteins produced in various tissues after hyperthermia may be involved in homeostatic control in the heat-shock response of the rat.

Inhibition by rifamycin AF/013 of thyroid hormone binding to the nuclear receptor.

Rifamycin AF/013, a potent inhibitor of nucleic acid polymerizing enzymes and of some hormone receptors, strongly inhibited thyroid hormone-binding to the isolated nuclear receptor. Fifty percent inhibition was obtained at AF/013 concentration of as low as 8 micrograms/ml. AF/013, however, only weakly promoted dissociation of the bound hormone from the receptor. The inhibitory action of AF/013 was competitive with respect to and reduced the receptor's affinity for the hormone.

Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats.

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.

Presence of a unique set of nuclear proteins at the sites sensitive to RNase A as well as DNase I.

A unique and simple set of proteins (S1 proteins) has been detected in the cell nuclei of various tissues of the rat (Inoue et al. (1983) Eur. J. Biochem. 135, 61-68). They were liberated from the nuclei by digestion with DNA-hydrolyzing enzymes under the conditions where transcriptionally active chromatin is preferentially digested and released into the reaction supernatant. S1 proteins account for 20% of the total supernatant proteins (Higashi et al. (1984) Biochem. Int. 9, 697-704). The present study demonstrates that S1 proteins occur at the sites sensitive to RNase A as well as DNase I, and are rapidly liberated from the nuclei by digestion with either enzyme.

Calcitonin-induced phosphorylation of rat liver cytosolic proteins.

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.

Polyenthesitis.

We described the clinical symptoms and signs of 11 cases of polyenthesitis, confirmed by the presence of inflammation in biopsies and increased activity in scintigraphy. All cases are negative for HLA-B27, and there is no presence of systemic inflammatory reaction, radiographic sacroiliitis, or chronic arthritis. The enthesitis is not ossifying and follows a rather benign clinical course. In one case, herpes virus-like particles were detected in the fibroblast debris of the tissue removed from Achilles tendon insertion. This fact suggests that polyenthesitis may be a clinical manifestation due to a reaction to an infectious agent.