Effects of bisphosphonates on human esophageal squamous cell carcinoma cell survival.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients.

Tunable terahertz emission from the intrinsic Josephson junctions in acute isosceles triangular Bi2Sr2CaCu2O8+δ mesas.

In order to determine if the mesa geometry might affect the properties of the coherent terahertz (THz) radiation emitted from the intrinsic Josephson junctions in mesas constructed from single crystals of the high-temperature superconductor, Bi2Sr2CaCu2O8+δ, we studied triangular mesas. For equilateral triangular mesas, the observed emission was found to be limited to the single mesa TM(1,0) mode. However, tunable radiation over the range from 0.495 to 0.934 THz was found to arise from an acute isosceles triangular mesa. This 47% tunability is the widest yet observed from the outer current-voltage characteristic branch of such mesas of any geometry. Although the radiation at a few of the frequencies in the tunable range appear to have been enhanced by cavity resonances, most frequencies are far from such resonance frequencies, and can only be attributed to the ac-Josephson effect.

Fibroblast growth enhancing activity of tumor necrosis factor and its relationship to other polypeptide growth factors.

Tumor necrosis factor (TNF) is a monocyte-derived protein cytotoxic or cytostatic for some tumor cell lines. Here we show that highly purified E. coli-derived recombinant human TNF stimulated the growth of human FS-4 diploid fibroblasts. Stimulation of cell growth was demonstrable at a TNF concentration of 10 pg/ml (3 X 10(-13) M). Maximal stimulation was attained at TNF concentrations of 10 ng/ml (3 X 10(-10) M) or higher. Growth-stimulatory activity of TNF was inhibited by an mAb neutralizing the cytotoxic activity of TNF. Growth stimulation was not inhibited by another mAb specific for TNF, lacking neutralizing activity for the cytotoxic activity of TNF. Growth stimulation by TNF was more marked and more sustained in the presence of greater than or equal to 10% FCS than in medium with less than or equal to 5% FCS. Addition of TNF to confluent FS-4 cultures also produced a marked stimulation of cell growth in the presence of fresh FCS, while a much less marked stimulation was seen in the absence of FCS. Stimulation of confluent cultures by TNF in serum-free medium was enhanced by insulin, suggesting that insulin or insulin-like growth factor(s) in the serum can act synergistically with TNF in producing growth stimulation. While the growth-stimulatory effects of TNF and insulin were synergistic, the actions of TNF and epidermal growth factor (EGF) were less than additive, suggesting that TNF and EGF may activate identical or similar pathways. We conclude that stimulation of cell growth is probably a physiological function of TNF, and that the cytotoxic and cytostatic actions of TNF may be the result of an anomalous growth signal transduction in neoplastic cells lacking the constraints of normal growth control mechanisms.

Pharmacokinetic modeling of the dosing interval dependency for the interaction between itraconazole and triazolam.

OBJECTIVE: Itraconazole is a potent inhibitor of cytochrome P450 (CYP) 3A with an elimination half-life of more than 30 hours. Therefore, itraconazole may cause persistent CYP3A inhibition. Triazolam is primarily metabolized by CYP3A and its plasma concentration is increased remarkably by itraconazole. Although separating their dosages by 24 hours has been shown to reduce the interaction, an appropriate dosage interval remains to be determined. The aim of this study was to identify an appropriate dosage schedule to avoid their interaction. MATERIALS AND METHODS: We developed a pharmacokinetic model based on the assumption that both itraconazole and hydroxyitraconazole competitively and reversibly inhibit the first-pass metabolism and systemic elimination of triazolam. The developed model was simultaneously fitted to the plasma concentration profiles of triazolam, taken from the literature, by using the plasma concentration-time profiles of itraconazole and hydroxyitraconazole as input functions to estimate their in vivo Ki values. Subsequently, we simulated the plasma concentration profiles of triazolam administered after itraconazole therapy with various dosing intervals. RESULTS: The model could explain and simulate the interaction between itraconazole-triazolam using a variety of dosage intervals between the administrations. CONCLUSIONS: The developed model may provide useful information with regard to the appropriate interval for triazolam administration during itraconazole therapy.

X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer.

The x-ray structure of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine-dimer-specific excision-repair pathway, was determined at a 1.6-angstrom resolution. The enzyme consists of a single compact domain classified into an all-alpha structure. This single domain has two distinct catalytic activities; it functions as a pyrimidine dimer glycosylase and as an apurinic-apyrimidinic endonuclease. The amino-terminal segment penetrates between two major helices and prevents their direct contact. The refined structure suggests the residues involved in the substrate binding and the catalysis of the glycosylation reaction.

Sperm chromatin decondensation by template activating factor I through direct interaction with basic proteins.

Template activating factor I (TAF-I) was originally identified as a host factor required for DNA replication and transcription of adenovirus genome complexed with viral basic proteins. Purified TAF-I was shown to bind to core histones and stimulate transcription from nucleosomal templates. Human TAF-I consists of two acidic proteins, TAF-Ialpha and TAF-Ibeta, which differ from each other only in their amino-terminal regions. Here, we report that TAF-I decondenses demembraned Xenopus sperm chromatin. Human TAF-Ibeta has a chromatin decondensation activity comparable to that of NAP-I, another histone binding protein, whereas TAF-Ialpha has only a weak activity. Analysis of molecular mechanisms underlying the chromatin decondensation by TAF-I revealed that TAF-I interacts directly with sperm basic proteins. Deletion of the TAF-I carboxyl-terminal acidic region abolishes the decondensation activity. Interestingly, the acidic region itself is not sufficient for decondensation, since an amino acid substitution mutant in the dimerization domain of TAF-I which has the intact acidic region does not support chromatin decondensation. We detected the beta form of TAF-I in Xenopus oocytes and eggs by immunoblotting, and the cloning of its cDNA led us to conclude that Xenopus TAF-Ibeta also decondenses sperm chromatin. These results suggest that TAF-I plays a role in remodeling higher-order chromatin structure as well as nucleosomal structure through direct interaction with chromatin basic proteins.

Antacid interaction with new quinolones: dose regimen recommendations based on pharmacokinetic modeling of clinical data for ciprofloxacin, gatifloxacin and norfloxacin and metal cations.

OBJECTIVE: New quinolones (NQs) are widely used to treat various infections. However, concomitant oral administration of metal cations may decrease absorption of NQs and consequently decrease their blood concentration and pharmacological effect. A convenient approach to avoid this interaction is to separate the dosages by a certain interval. In this study, we aimed to develop a novel pharmacokinetic model to describe NQs-metal cation interactions in order to estimate the optimal dosing interval. METHODS: Plasma concentration-time profiles of NQs after administration without or with metal cations at various dosing intervals were collected from the literature and analyzed with a pharmacokinetic model incorporating the formation ofNQs-metal cations complex. The model was fitted to the reported time profiles ofciprofloxacin (CPFX) plasma concentration after concomitant administration with aluminum hydroxide/magnesium hydroxide antacid (Al/Mg antacid; Maalox, Maalox70) at various dosing intervals to obtain the pharmacokinetic parameters of CPFX. Model analysis was also carried out for gatifloxacin (GFLX) and norfloxacin (NFLX). RESULTS: The developed model could adequately explain the interactions in all the combinations investigated. The model predicted, in the cases of usual doses of CPFX with Maalox, GFLX with Maalox70 and NFLX with sucralfate, that the NQ should be administered 4.5, 4.5 and 3.5 hours after, or 1, 1 and 0.5 hours before the administration of metal cations, respectively, to ensure 90% of control absorption. CONCLUSIONS: The developed model can adequately describe the extent of interaction between NQs and metal cations, and should be clinically useful to design dosage regimens to circumvent the interaction.

A clinicopathologic study of p27(kip1) expression in renal allograft biopsy.

BACKGROUND: P27 (Kip1) is an inhibitor of cyclin-dependent kinases/cyclin complex that keeps mature cells growth-arrested. In IgA nephropathy, a decreased p27kip1 expression in podocytes has been reported to be related to lesion formation of focal segmental glomerulosclerosis and renal dysfunction. We reviewed the p27kip1 expression in transplanted kidneys. METHODS: p27kip1 expression was examined immunohistochemically in 26 allograft biopsy specimens. RESULTS: p27kip1 expression was recognized in podocytes. Patients with more than 0.5 g proteinuria showed fewer p27kip1-positive cells than those with less than 0.5 g proteinuria. The decreased p27kip1 expression in podocytes was related to cg and ah of the Banff 97 classification. In the two cases in which p27kip1 expression was remarkably decreased, elevation of the serum creatinine level was recognized at the time of biopsy, resulting in kidney transplant loss. The histological findings were chronic/sclerosing allograft nephropathy grade II-(b) in both cases. CONCLUSION: In conclusion, decreased p27kip1 expression in podocytes suggested a significant role in proteinuria among renal transplant recipients.

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

Importance of lymph vessels in gastric cancer: a prognostic indicator in general and a predictor for lymph node metastasis in early stage cancer.

BACKGROUND: Metastasis to regional lymph nodes (LNs) through lymphatic vessels is common in cancer progression and is an important prognostic factor in many cancers. Recent evidence suggests that tumour lymphangiogenesis promotes lymphatic metastasis. AIMS: To study the role of lymph vessel density (LVD) in gastric cancer and investigate whether LVD is associated with LN metastasis/prognosis. METHODS: Lymphatics of 117 primary human gastric cancer cases were investigated by quantitative immunohistochemical staining for podoplanin. The relation between LVD and LN metastasis and other established clinicopathological parameters was analysed. The relation between LVD and prognosis was also studied. RESULTS: Mean LVD of "hot spots" was 11.6/case. LVD significantly correlated with LN and podoplanin positive lymphatic invasion. High LVD was associated with worse overall survival. In multivariate analysis, positive LVD was a significant independent predictor of overall survival, depth of invasion, and TNM stage. LVD significantly correlated with LN metastasis at surgery and podoplanin positive lymphatic invasion. In multivariate analysis, positive LVD was an independent significant predictor of LN metastasis. CONCLUSIONS: Increased podoplanin expression is significantly associated with LN metastasis, and may play an important role in detecting LN metastasis in gastric cancer. Furthermore, LVD may be a significant prognostic factor in gastric cancer at any stage. In addition, LVD and lymph vessel invasion detected by podoplanin immunohistochemistry are associated with LN metastasis in T1 early gastric cancer. LVD assessment by podoplanin immunohistochemistry may become a useful predictor of LN metastasis in T1 early gastric cancer and may influence the decision making process for additional surgery.

Dosage adjustment of quinolone antibiotics and angiotensin-converting enzyme inhibitors in patients with renal dysfunction.

The aim of this study was to verify an approach for calculating pharmacokinetic parameters suitable for adjusting dosage regimens in patients with renal dysfunction. We carried out a retrospective analysis of the pharmacokinetic profiles of 12 new quinolone antibiotics and 11 angiotensin-converting enzyme inhibitors (ACEIs) in patients with normal and impaired renal function to obtain the renal excretion ratio (R(renal)) of each drug. We demonstrated that the pharmacokinetics of each drug in a patient with renal dysfunction can be adequately estimated using the R(renaI) value of each drug together with the creatinine clearance as an index of the individual's renal function. Using the R(renaI) value obtained, we could successfully simulate pharmacokinetic profiles of the drugs in publications other than that used to obtain the R(renal) values. On the other hand, age-related changes in the pharmacokinetics of new quinolone antibiotics are not always adequately predicted using the R(renal) value compared to using creatinine clearance alone as an index, and the reasons for this are not fully understood. These results demonstrate that dosage regimens of quinolone antibiotics and ACEIs in patients with renal dysfunction can be adequately optimized using the R(renal) value for each drug using the present approach.

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes.

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)-cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.

Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors.

The pathogenesis of carcinosarcoma is still a subject of controversy. In the present study, molecular techniques were applied to determine the pathogenesis of uterine carcinosarcomas. The patterns of chromosome X inactivation were analyzed, targeting a portion of exon 1 of the human androgen receptor (HUMARA) in malignant epithelial and mesenchymal components. The presence of p53 and K-ras mutations were also analyzed. H&E-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both epithelial and nonepithelial lesions from 25 carcinosarcomas, and DNAs were extracted by proteinase K digestion. Following treatment with methylation-sensitive restriction endonuclease (HhaI or HpaII), PCR amplification was performed using nested primers targeted to the HUMARA locus. Mutations in the p53 gene and K-ras gene were found in eight (32%) and six (24%) tumors, respectively. The patterns of chromosome X inactivation were different between the carcinomatous and sarcomatous components of three carcinosarcomas, indicating that these three tumors represent collision tumors. By contrast, the patterns of chromosome X inactivation, K-ras sequence, and p53 sequence were identical in both carcinomatous and sarcomatous components in 21 carcinosarcomas, indicating that these 21 tumors represent combination tumors. One case produced equivocal results that precluded determination of whether it represented a collision or combination tumor. These observations show that although most carcinosarcomas are combination tumors, some develop as collision tumors. The determination of histogenesis in individual cases of carcinosarcoma using molecular markers may be worthwhile, because the result could help predict the prognosis of individual cases and help guide clinical management.

Calibration of the microcalorimeter spectrometer on-board the Hitomi (Astro-H) observatory (invited).

The Hitomi Soft X-ray Spectrometer (SXS) was a pioneering non-dispersive imaging x-ray spectrometer with 5 eV FWHM energy resolution, consisting of an array of 36 silicon-thermistor microcalorimeters at the focus of a high-throughput soft x-ray telescope. The instrument enabled astrophysical plasma diagnostics in the 0.3-12 keV band. We introduce the SXS calibration strategy and corresponding ground calibration measurements that took place from 2012-2015, including both the characterization of the microcalorimeter array and measurements of the x-ray transmission of optical blocking filters.

Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells.

Three cDNA clones of rat renal dipeptidase (rrDP) were isolated from rat renal and pulmonary cDNA libraries using a DNA fragment of human renal DP cDNA clone, MDP4, as a probe. The complete amino acid sequence deduced from the cDNA contains 410 amino acid residues, beginning with a signal peptide of 16 amino acid residues. RNA blot hybridization analysis showed that 1.6 and 2.2 kb mRNAs were expressed in lung and kidney, however, only 1.6 kb mRNA was detected in small intestine. COS-1 cells transfected with the cDNA expressed enzymatically active rrDP.

Importance of Glu-125 in the catalytic activity of human renal dipeptidase.

The carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and dansyl-ethylenediamine have shown to inhibit human renal dipeptidase (hrDP) irreversibly in a time-dependent manner. Cilastatin, a competitive inhibitor of the enzyme, partially protected the enzyme from inactivation. To identify the site(s) modified by EEDQ and dansylethylenediamine, the amino-acid sequence of tryptic fragments of modified enzyme were analyzed extensively. A comparison of the determined amino-acid sequences with the predicted primary structure of hrDP revealed that Glu-125 within the Glu115-Arg138 fragment was modified. In consequence, the role of Glu-125 in catalytic activity was investigated by site-directed mutagenesis. Glu-125 was replaced by a glutamine, aspartic acid or cysteine residue. cDNAs for wild-type or mutated enzymes were expressed in CHO cells, and the resulting proteins were purified to apparent homogeneity. The mutated enzyme, Gln-125-hrDP exhibited specific activity of 28.5 U/mg, corresponding to 11.4% of the wild-type. In contrast, Asp-125-hrDP and Cys-125hrDP were found to be inactive (< or = 0.1% of wild-type enzyme). These results suggest that the polarity and/or length of the side chain of Glu-125 residue are important for the enzyme activity.

Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain.

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.

Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins.

The processes governing chromatin remodeling and assembly, which occur prior to and/or after transcription and replication, are not completely understood. To understand the mechanisms of transcription and replication from chromatin templates, we have established in vitro replication and transcription systems using adenovirus (Ad) DNA complexed with viral basic core proteins, called Ad core, as a template. Using this system, we have previously identified, from HeLa cells, template activating factor-I as a stimulatory factor for the Ad core DNA replication. Here, using this system as a tool, we identified and purified a novel template activating factor activity that consists of two acidic polypeptides whose apparent molecular masses are 38 kDa and 37 kDa. These two polypeptides correspond to two splicing variants of nucleolar phosphoprotein, nucleophosmin/B23. Recombinant B23 proteins stimulate the Ad core DNA replication, and the acidic regions of B23 proteins are important for its activity. In addition, B23 proteins directly bind to core histones and transfer them to naked DNA. Furthermore, chromatin components such as histones and topoisomerase II are co-immunoprecipitated with B23 from cell extracts. These observations lead to a hypothesis that nucleophosmin/B23 is involved in structural changes of chromatin, thereby regulating transcription and replication within the ribosomal DNA region or maintaining the nucleolar structure.

Preliminary crystallographic study of pyrimidine dimer-specific excision-repair enzyme from bacteriophage T4.

Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2(1), with unit cell parameters: a = 41.4 A, b = 40.1 A, c = 37.5 A, beta = 90.01 degrees. The unit cell contains two 16,000 Mr molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.