Prior treatment with vitamin K(2) significantly improves the efficacy of risedronate.

UNLABELLED: Prior 8-week treatment with menatetrenone, MK-4, followed by 8-week risedronate prevented the shortcomings of individual drugs and significantly increased the strength of ovariectomized ICR mouse femur compared to the ovariectomized (OVX) controls. Neither MK-4 following risedronate nor the concomitant administration may be recommended because they brought the least beneficial effect. INTRODUCTION: The objective of this study was to determine the best combinatory administration of risedronate at 0.25 mg/kg/day (R) with vitamin K(2) at approximately 100 microg MK-4/kg/day (K) to improve strength of osteoporotic mouse bone. METHODS: Thirteen-week-old ICR mice, ovariectomized at 9-week, were treated for 8 weeks with R, K, or R plus K (R/K), and then, either the treatment was withdrawn (WO) or switched to K or R in the case of R and K. After another 8 weeks, the mice were killed, and mechanical tests and analyses of femur properties by peripheral quantitative computed tomography, microfocus X-ray tube computed tomography, and confocal laser Raman microspectroscopy were carried out. RESULTS: The K to R femur turned out superior in parameters tested such as material properties, bone mineral density, BMC, trabecular structure, and geometry of the cortex. The increased cross-sectional moment of inertia, which occurred after K withdrawal, was prevented by risedronate in K to R. In addition to K to R, some properties of R to WO diaphysis and K to WO epiphysis were significantly better than OVX controls. CONCLUSION: Prior treatment with MK-4 followed by risedronate significantly increased femur strength in comparison to the OVX controls.

Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line.

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by approximately 30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Differentiation inducers modulate cytokine signaling pathways in a murine erythroleukemia cell line.

Hexamethylenebisacetamide (HMBA) is a potent differentiation inducer of murine erythroleukemia cells. Immunoprecipitation followed by Western blotting with an anti-phosphotyrosine (P-Tyr) antibody revealed that HMBA increased P-Tyr levels and/or amounts of several proteins containing P-Tyr in F5-5, a murine erythroleukemia cell line. Among these proteins, we identified a Mr 130,000 protein to be Janus-activated kinase 2 (JAK2). HMBA induced tyrosine phosphorylation of JAK2 and signal transducers and activators of transcription 5 (STAT5) but not other JAKs or STATs. This phosphorylation was apparent 12 h after treatment, maximal at 24 h, and persisted for at least 96 h. Consistently, HMBA increased STAT5 DNA-binding activities. Other chemical inducers, DMSO and butyrate, also induced a sustained activation of JAK2/STAT5, whereas fetal calf serum and erythropoietin induced transient activation but not differentiation. Furthermore, overexpression of a dominant-negative form of STAT5 inhibited the chemically induced differentiation. These results suggest that persistent activation of the signaling pathway plays a significant role in the inducer-mediated differentiation. Our data also suggest that molecular mechanisms for the inducer-mediated activation of JAK2 are independent of cytokine receptor-mediated activation mechanisms. We tentatively conclude that cytokine signaling is an important target of chemical inducers in these cells.

Human T-cell leukemia virus type 1 Tax protein induces the expression of STAT1 and STAT5 genes in T-cells.

Human T-cell leukemia virus type 1 (HTLV-1) Tax transforms normal T-cells in the presence of interleukin (IL)-2 in vitro. STAT is a family of transcription factors that play a pivotal role in cytokine-induced functions of a various type of cells. We investigated the involvement of STATs in the transformation of T-cells by HTLV-1. HTLV-1-transformed T-cell lines expressed higher amounts of STAT1, STAT3 and STAT5 RNA and proteins than virus-negative T cells. The expression of STAT1 and STAT5 in a human T-cell line was induced by Tax. IL-2 induced the DNA binding activity of STAT3 and STAT5 of a HTLV-1-transformed cell line and then stimulated its proliferation. In contrast, IL-2 did neither in a cell line lacking STAT3 and STAT5. The expression of STAT1, STAT3 and STAT5 mRNAs were also induced by a T-cell mitogen in normal human peripheral blood mononuclear cells. Our results suggest that the induction of STAT1 and STAT5 by Tax enhances cytokine-induced functions of virus-infected T-cells, hence the induction may play a role in IL-2-dependent transformation steps of T-cells by HTLV-1.

cDNA cloning and expression analysis of mouse zf9, a Krüppel-like transcription factor gene that is induced by adipogenic hormonal stimulation in 3T3-L1 cells.

Using a differential hybridization method, we have cloned a zinc finger transcription factor gene whose expression was enhanced by adipogenic hormones in preadipocyte 3T3-L1 cells. Cloning of this gene revealed that it encodes a mouse homologue of rat Zf9 and human CPBP/GBF, previously identified as a wound-induced transcription factor and GC-rich binding protein, respectively. The mRNA for this clone consisted of 0.9 kb coding region and 3.2 kb long 3' untranslated region. Northern blot analysis revealed that it was ubiquitously expressed, among adult tissues, in which abundant expression was observed in lung, ovary and thymus. The transcript was transiently induced by different stimuli such as serum, cAMP and 12-O-tetradecanoylphorbol 13-acetate. Nuclear run-on and RNA synthesis inhibitor chase experiments indicated that the transient induction of the mRNA was regulated both at transcriptional and post-transcriptional levels.

The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex.

The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm. In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion. All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

The role of erythropoietin receptor tyrosine phosphorylation in erythropoietin-induced proliferation.

Although studies with truncated erythropoietin receptors (EpoRs) have suggested the tyrosine phosphorylation (Yphos) of the EpoR may not play a significant role in Epo-induced proliferation, we found, using a full length EpoR mutant designed Null, in which all 8 of the intracellular tyrosines (Ys) were substituted with phenylalanines (Fs), that Null cells required 5-10 fold more Epo than wild type (WT) EpoR containing cells in order to proliferate as well. Moreover, a comparison of Epo-induced proliferation with Epo-induced Yphos patterns, using DA-3 cells expressing WT, Null and various Y to F EpoR point mutants revealed that Stat5 Yphos and activation correlated directly with proliferation and was mediated primarily throuhg the most membrane proximal Y, i.e., Y343, although other tyrosines (most likely Y401 and Y431) within the EpoR could activate Stat5 in its absence. We also found that EpoR Yphos was essential for the Yphos of Shc and for the Yphos and association of a 145 kDa protein with Shc. We purified and cloned this Shc-associated 145 kDa protein and found that it was a unique SH2 containing inositol polyphosphate-5-phosphatase. This novel enzyme, which we have called SHIP for SH2-containing inositol-phosphatase, may modulate both Ras and inositol signaling pathways.

Transcriptional regulation of the beta-casein gene by cytokines: cross-talk between STAT5 and other signaling molecules.

The beta-casein promoter has been widely used to monitor the activation of STAT (signal transducer and activator of transcription)5 since STAT5 was originally found as a mediator of PRL-inducible beta-casein expression. However, not only is expression of the beta-casein gene regulated by STAT5 but it is also affected by other molecules such as glucocorticoid and Ras. In this report, we describe the transcriptional regulation of the beta-casein gene by cytokines in T cells. We have found that the beta-casein gene is expressed in a cytotoxic T cell line, CTLL-2, in response to interleukin-2 (IL-2), which activates STAT5. While IL-4 does not activate STAT5, it induces expression of STAT5-regulated genes in CTLL-2, i.e. beta-casein, a cytokine-inducible SH2-containing protein (CIS), and oncostatin M (OSM), suggesting that STAT6 activated by IL-4 substitutes for the function of STAT5 in T cells. IL-2-induced beta-casein expression was enhanced by dexamethasone, and this synergistic effect of Dexamethasone requires the sequence between -155 and -193 in the beta-casein promoter. Coincidentally, a deletion of this region enhanced the IL-2-induced expression of beta-casein. Expression of an active form of Ras, Ras(G12V), suppressed the IL-2-induced beta-casein and OSM gene expression, and the negative effect of Ras is mediated by the region between -105 and -193 in the beta-casein promoter. In apparent contradiction, expression of a dominant negative form of Ras, RasN17, also inhibited IL-2-induced activation of the promoter containing the minimal beta-casein STAT5 element as well as the promoters of CIS and OSM. In addition, Ras(G12V) complemented signaling by an erythropoietin receptor mutant defective in Ras activation and augmented the activation of the beta-casein promoter by the mutant erythropoietin receptor signaling, suggesting a possible role of Ras in Stat5-mediated gene expression. These results collectively reveal a complex interaction of STAT5 with other signaling pathways and illustrate that regulation of gene expression requires integration of opposing signals.

Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells.

Extracellular stimuli trigger adipocyte differentiation by inducing the complex cascades of transcription. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma (PPARgamma) play crucial roles in this process. Although ectopic expression of these factors in NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in adipogenic conversion, little is known as to hormonal factors that regulate adipogenesis in these cells. In this report we demonstrate that PRL, a lactogenic hormone, enhances C/EBPbeta and PPARbeta mRNA expression and augments adipogenic conversion of NIH-3T3 cells. Moreover, we show that ectopic expression of the PRL receptor in NIH-3T3 cells results in efficient adipocyte conversion when stimulated with PRL and a PPARgamma ligand, as evidenced by expression of the adipocyte differentiation-specific genes as well as the presence of fat-laden cells. We further demonstrate that signal transducer and activator of transcription 5 (Stat5), a PRL signal transducer, activates aP2 promoter in a PRL-dependent manner. These results suggest that PRL acts as an adipogenesis-enhancing hormone in NIH-3T3 cells.

Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of STAT5.

Recently, JAK2 kinase was found to be one of the tyrosine kinases activated by interleukin-3 (IL-3) in target cells. JAK2 belongs to a family of kinases that act upstream of transcription factors called STATs. STATs exist in the cytoplasm as latent, transcriptionally inactive forms until, in response to extracellular signals, they become phosphorylated on tyrosine residues, translocate to the nucleus, and bind to specific DNA elements. Because IL-3 activates JAK2, we searched for the STAT(s) that might transduce IL-3 signals. Several lines of evidence suggest that IL-3 uses the murine homologue of STAT5, a factor originally purified from sheep. Unexpectedly, during isolation of the murine homologue, we found two highly related molecules that we have designated STAT5A and STAT5B.

Human SOX11, an upregulated gene during the neural differentiation, has a long 3' untranslated region.

To obtain essential genes for neuronal development, we have performed a molecular indexing method using a human teratocarcinoma cell line, NTera-2. We isolated a cDNA fragment, designated B18, as an upregulated gene during the neural differentiation. From the complete cDNA sequence of B18 it was revealed that this cDNA was the human SOX11 gene. While a previous report has determined only a approximately 2 kb of the SOX11 cDNA including the entire open reading frame, our full length cDNA was 8743 bp possessing a long 3' untranslated region. Human SOX11 cDNA was mapped to chromosome region 2p25.3 between markers AFMA070WC9 and WI-1412 by radiation hybrid mapping.

Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex.

The specific effect of the binding of initiation factor IF2 on E coli 16S rRNA within the [IF2/30S/GTP] complex has been probed by crosslinking experiment with trans-diamminedichloro platinum (II) and by phosphate alkylation with ethylnitrosourea. Several 16S rRNA fragments crosslinked to IF2 have been identified and are mostly located in the head and the lateral protrusion of the 30S subunit. The study of the effect of IF2 binding to the 30S subunit reveals that the factor does not tightly bind to the 16S rRNA and induces both isolated reductions and enhancements of phosphate reactivity in the 16S rRNA. Several of them are located near the binding site of IF2 and weak effects are observed in distant parts of the subunit. These results are discussed in the light of current knowledge of the topographical localization of IF2 with the 30S subunit and of its relation with function.

Quantitative gallium-67 scanning for predictive value in primary lung carcinoma.

Gallium-67 scintigraphy was performed on 87 patients with a variety of histological types of untreated primary lung carcinoma. Gallium-67 uptake was determined, allowing for differences in tumor size. Differential uptakes were found for the various tumor types, with anaplastic small-cell carcinoma having the greatest average uptake, and adenocarcinoma and anaplastic large-cell carcinoma the smallest. Gallium-67 uptake was compared with response to radiation therapy, incidence of metastasis, and host survival in 58 of the patients. From these results it is suggested that the greater the Ga-67 accumulation in the tumor, the more effective is radiation therapy in reducing tumor size. Gallium-67 scintigraphy appears to be a valuable tool in estimating the sensitivity of the tumor before radiation therapy and in indicating the prognosis following radiation therapy in patients with primary lung carcinoma.

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius.

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.

Bone scintigraphy in detection of bone invasion by oral carcinoma.

Detecting osseous involvement is clinically important in the management of oral carcinoma. Thirty-one patients with osseous involvement due to oral carcinoma who underwent panoramic radiography and bone scintigraphy were evaluated retrospectively. Bone scintigraphy confirmed osseous involvement in all 31 (100%) of these patients. In 27 (87%) of 31 patients with osseous involvement, both the panoramic radiogram and bone scintigram were positive. In the remaining four patients (13%), bone scintigram was positive for mandibular or maxillary invasion, while panoramic radiogram was negative. There were no instances of an abnormal radiogram with a normal bone scintigram. These findings strongly suggest that bone scintigraphy is more sensitive than panoramic radiography in detecting osseous involvement of the mandible and maxilla due to oral carcinoma. Furthermore, bone scintigraphy was a critical pre-surgical in determining the extent of the osseous involvement.

Ga-67 scan as a prognostic indicator in primary lung carcinoma.

GA-67 scintigraphy was performed on 74 patients with a variety of histologic types of untreated primary lung carcinoma. Ga-67 uptake was determined, allowing for differences in tumor size. Ga-67 uptake was compared with the response to the incidence of metastases, and to host survival in the 74 patients. From these results, it is suggested that the greater the Ga-67 accumulation in the tumor, the higher the incidence of metastases and the shorter the host survival. Ga-67 scintigraphy appears to be a valuable tool in indicating the prognosis following radiation therapy in patients with primary lung carcinoma.

Technetium-99m pertechnetate and gallium-67 imaging in salivary gland disease.

Thirty-two patients with salivary gland tumors or sialadenitis were studied with Tc-99m pertechnetate and Ga-67 imaging and, in some instances, sialography. The diagnostic algorithm presented allows the correct categorization of the salivary gland pathology in the vast majority of patients. The patients were studied serially with Tc-99m pertechnetate, Ga-67 and in certain situations sialography (or CT-sialography). Use of the algorithm can distinguish benign salivary tumors from malignant tumors and malignant tumors from inflammatory disease. The limitations and pitfalls of interpretation are discussed.

[Simplified analytical method of 99mTc-labeled phosphonate].

A simplified and rapid analytical method of 99mTc-labeled phosphonates was tested using a mini-column based on an anion-exchange type of cartridge. Free 99mTcO4- in the prepared solutions of 99mTc-labeled phosphonate was eluted from the column by a neutral phosphate buffer solution. Partly components of the 99mTc-labeled phosphonates was eluted from the column by a 100 mM sodium phosphonate solution, while the residual components were not eluted from the mini-column. In addition, for analysis of 99mTc-labeling rate in 99mTc-MDP solution, this method requires much less time than thin layer chromatography (TLC). Therefore, the method is more suitable for analysis of 99mTc-labeled phosphonates than TLC now in use, particularly rapid analysis for 99mTc-labeling rate of the compounds and the stability.

[Development of helical CT and clinical application].

We applied helical CT to examinations of the clinical image diagnosis. Scanning was performed continuously while the couchtop of the CT scanner was shifted at a constant speed. The entire lung field could be scanned during a single holding of the breath when the couchtop speed was 20 mm/s. Tumors as small as 2 mm were detected. The diagnostic detectability and accuracy of helical CT were far superior to those conventional chest radiography. During abdominal examination, the target organ could be scanned at any specified phase (preferably arterial) during the injection of contrast medium. We detected very small hepatic tumors which could not be detected by conventional CT. Helical CT produced continuous data, which was reconstructed to display three-dimensional image. Helical CT could be used in mass screening for the detection of early lung cancer and in computer-aided diagnosis.