Outcome of maintenance systemic chemotherapy with drug-free interval for metastatic urothelial carcinoma.

OBJECTIVE: Aiming to achieve long-term disease control, maintenance systemic chemotherapy (MSC) with a 1-3-month drug-free interval is continued in selected patients. We report our experience of MSC for metastatic urothelial carcinoma (UC). METHODS: Of 228 metastatic UC patients treated with systemic chemotherapy, 40 (17.5%, 40/228) had continuously undergone MSC. Data on the regimen, cycle number, and reason for the discontinuation of MSC were also collected. We analyzed OS from the initiation of MSC until death or the last follow-up, using the log-rank test to assess the significance of differences. RESULTS: The median number of cycles of chemotherapy was 6, and the responses were CR in 6, PR in 20, SD in 13, and PD in 1 before MSC. Gemcitabine plus CDDP or carboplatin was mainly performed as MSC (70%, 28/40). MSC was repeated quarterly in 30 (75%, 30/40), every two months in 8 (20%, 8/40), and with other intervals in 2 (5%, 2/40). Overall, a median of 3.5 cycles (range: 1-29) of MSC was performed. The reason for the discontinuation of MSC was PD in 24 (60%, 24/40), favorable disease control in 9 (22.5%, 9/40), and myelosuppression in 3 (7.5%, 3/40), and for other reasons in 2 (5%, 2/40). MSC was ongoing in 2 (5%, 2/40). The median OS was 27 months from the initiation of MSC. PS0 (P = 0.0169), the absence of lung metastasis (P = 0.0387), and resection of the primary site (P = 0.0495) were associated with long-term survival after MSC. CONCLUSIONS: In selected patients, long-term systemic chemotherapy could be performed with a drug-free interval. Our maintenance strategy with cytotoxic drugs may become one of the treatment options for long-term disease control.

Generation of pressures over 40 GPa using Kawai-type multi-anvil press with tungsten carbide anvils.

We have generated over 40 GPa pressures, namely, 43 and 44 GPa, at ambient temperature and 2000 K, respectively, using Kawai-type multi-anvil presses (KMAP) with tungsten carbide anvils for the first time. These high-pressure generations were achieved by combining the following pressure-generation techniques: (1) precisely aligned guide block systems, (2) high hardness of tungsten carbide, (3) tapering of second-stage anvil faces, (4) materials with high bulk modulus in a high-pressure cell, and (5) high heating efficiency.

Combining FIB milling and conventional Argon ion milling techniques to prepare high-quality site-specific TEM samples for quantitative EELS analysis of oxygen in molten iron.

This paper reports a procedure to combine the focused ion beam micro-sampling method with conventional Ar-milling to prepare high-quality site-specific transmission electron microscopy cross-section samples. The advantage is to enable chemical and structural evaluations of oxygen dissolved in a molten iron sample to be made after quenching and recovery from high-pressure experiments in a laser-heated diamond anvil cell. The evaluations were performed by using electron energy-loss spectroscopy and high-resolution transmission electron microscopy. The high signal to noise ratios of electron energy-loss spectroscopy core-loss spectra from the transmission electron microscopy thin foil, re-thinned down to 40 nm in thickness by conventional Argon ion milling, provided us with oxygen quantitative analyses of the quenched molten iron phase. In addition, we could obtain lattice-fringe images using high-resolution transmission electron microscopy. The electron energy-loss spectroscopy analysis of oxygen in Fe(0.94)O has been carried out with a relative accuracy of 2%, using an analytical procedure proposed for foils thinner than 80 nm. Oxygen K-edge energy-loss near-edge structure also allows us to identify the specific phase that results from quenching and its electronic structure by the technique of fingerprinting of the spectrum with reference spectra in the Fe-O system.

Superhard semiconducting optically transparent high pressure phase of boron.

An orthorhombic (space group Pnnm) boron phase was synthesized at pressures above 9 GPa and high temperature, and it was demonstrated to be stable at least up to 30 GPa. The structure, determined by single-crystal x-ray diffraction, consists of B12 icosahedra and B2 dumbbells. The charge density distribution obtained from experimental data and ab initio calculations suggests covalent chemical bonding in this phase. Strong covalent interatomic interactions explain the low compressibility value (bulk modulus is K300=227 GPa) and high hardness of high-pressure boron (Vickers hardness HV=58 GPa), after diamond the second hardest elemental material.

An insight into what superconducts in polycrystalline boron-doped diamonds based on investigations of microstructure.

The discovery of superconductivity in polycrystalline boron-doped diamond (BDD) synthesized under high pressure and high temperatures [Ekimov, et al. (2004) Nature 428:542-545] has raised a number of questions on the origin of the superconducting state. It was suggested that the heavy boron doping of diamond eventually leads to superconductivity. To justify such statements more detailed information on the microstructure of the composite materials and on the exact boron content in the diamond grains is needed. For that we used high-resolution transmission electron microscopy and electron energy loss spectroscopy. For the studied superconducting BDD samples synthesized at high pressures and high temperatures the diamond grain sizes are approximately 1-2 mum with a boron content between 0.2 (2) and 0.5 (1) at %. The grains are separated by 10- to 20-nm-thick layers and triangular-shaped pockets of predominantly (at least 95 at %) amorphous boron. These results render superconductivity caused by the heavy boron doping in diamond highly unlikely.

Exploratory aspirin resistance trial in healthy Japanese volunteers (J-ART) using platelet aggregation as a measure of thrombogenicity.

Aspirin prevents the production of thromboxane A2 (TXA2) by irreversibly inhibiting platelet cyclooxygenase, exhibiting antiplatelet actions. This agent has been reported to prevent relapse in patients with ischemic heart disease or cerebral infarction via this action mechanism. However, there are individual differences in this action, and aspirin is not effective in some patients, which is referred to as 'aspirin resistance'. In this study, we analyzed laboratory aspirin resistance by platelet aggregation in 110 healthy adult Japanese males using 24 single-nucleotide polymorphisms (SNPs) of nine genes involved in platelet aggregation/hemorrhage. Among SNPs involved in platelet aggregation, aspirin was less effective for 924T homozygote of a TXA2 receptor, 924T>C, and 1018C homozygote of a platelet membrane glycoprotein GPIbalpha, 1018C>T, suggesting that 924T and 1018C alleles are involved in aspirin resistance.

Phase transformations in bulk nanostructured potassium niobiosilicate glasses.

In potassium niobiosilicate (KNS) glasses, nanostructuring can be driven and controlled by thermal treatments at the glass transition temperature and/or by modulation of the chemical composition. The tight relationship between nanostructure and nonlinear optical properties suggests these bulk nanomaterials as an appealing route to nanophotonics. The focus of this paper is placed on assessing the phase transformations which occur in these materials upon annealing at the glass transition temperature and subsequent heating. High-temperature resolved X-ray diffraction (HTXRD) and high-resolution transmission electron microscopy (HRTEM) experiments are integrated with previously published results for in-depth insight. It will be shown that nanostructuring evolves from nucleation of niobium-rich nanocrystals, which are up to 20 nm large, uniformly distributed in the matrix bulk, and metastable. Formation kinetics as well as phase transformation of the nanocrystals are determined by the glass composition. Depending on it, nanocrystal nucleation can be preceded or not by phase separation, and the nanocrystals' phase transition can be of first or second order.

Splenic marginal zone lymphoma presenting as myelofibrosis associated with bone marrow involvement of lymphoma cells which secrete a large amount of TGF-beta.

We report here on a patient with splenic marginal zone lymphoma presenting diffuse fibrosis of bone marrow and spleen. After splenectomy and chemotherapy, bone marrow biopsy demonstrated an improvement of fibrosis. Plasma concentration of transforming growth factor (TGF)-beta was much higher in this patient than in those of age-matched non-Hodgkin's lymphoma patients ( n=5) at diagnosis, decreasing after resolution of myelofibrosis. Immunostaining with the TGF-beta antibody revealed that the lymphoma cells in bone marrow and spleen were positive for TGF-beta. TGF-beta secreted by tumor cells was thought to stimulate the growth of fibroblasts and synthesize collagen in bone marrow and splenic fibroblasts, and play an important role in the development of marrow and splenic fibrosis in this patient. This is the first report of a patient with splenic marginal zone lymphoma presenting as myelofibrosis associated with bone marrow involvement of lymphoma cells which secrete a large amount of TGF-beta.

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

[A case of lung tumorlets with pulmonary carcinoid].

It has been postulated that differential pathological diagnosis of lung tumorlet from pulmonary carcinoid is very important to decide the therapeutic strategy, because both of them are pathologically consisted of similar type of cells originated from same cells. At the present, although lung tumorlet is considered to be a hyperplastic lesion of Kultschitzky cells which is located in the epithelial cell of the bronchial mucosa by stimuli such as hypoxia and inflammation, but it occasionally recognized in the normal lung and the concept that it is a subtype of carcnoid is also undeniable. In this paper, a case of lung tumorlet with minute pulmonary carcinoid suggesting a subtype of carcinoid operated upon in our department is presented. Although simple pulmonary resection is a method of choice for pulmonary carcinoid, but it is still controversial as to therapeutic strategy for the lung tumorlet, because it is a benign entity and not tumorous lesion. However pulmonary resection with a close subsequent followed-up study must be a best method of choice for the case with lung tumorlet includes a minute lesion of carcinoid seen in this particular case.

cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region.

Members of the RAB protein family are important regulators of vesicular fusion and trafficking. A putative new member of the RAB family of genes was identified through a public database search, and its full-length cDNA was isolated from a human fetal brain cDNA library. The predicted protein product of the gene consists of 190 amino acid residues and has 87% identity with rat Rab26. Thus, we designated this gene as the human RAB26-related gene. Reverse transcription-coupled polymerase chain reaction (RT-PCR) demonstrated that the RAB26-related messenger RNA was predominantly expressed in adult and fetal brain. Furthermore, an RT-PCR experiment for brain subregions showed that the mRNA was highly expressed in the amygdala, cerebellum, caudate nucleus, and hippocampus. By PCR-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 16p13.3 region between markers WI-7742 and WI-3061. The RAB26-related gene consists of eight exons that span about 44kb of the genome DNA.

Isolation of a cDNA for a novel human RING finger protein gene, RNF18, by the virtual transcribed sequence (VTS) approach(1).

We have recently developed a novel database system, designated as the virtual transcribed sequence (VTS) which efficiently extracts many genes from public human genome databases, and tested the feasibility of this novel computational approach (N. Miyajima, C. Burge, T. Saito, Biochem. Biophys. Res. Commun. 272 (2000) 801; http://host45.maze.co.jp/vts/). In this study, using the VTS approach, we isolated a cDNA for a novel human gene with RING finger motif (C(3)HC(4)), which is not deposited in public EST databases. The isolated cDNA clone is 2163 bp in length, and contains an open reading frame of 452 amino acids. We designated the novel gene as RNF18. A database search showed that the RNF18 gene had the moderate similarity to SS-A/Ro52 protein, which is a ribonucleoprotein reactive with autoantibodies in patients with Sjögren's syndrome and systemic lupus erythematosus. Tissue distribution analyses by Northern blot and RT-PCR methods demonstrated that the RNF18 messenger RNA was preferentially expressed in testis. The exon-intron boundaries of RNF18 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The isolated cDNA consists of eight exons that span about 11 kb of the genome DNA. The precise chromosomal location of the RNF18 gene was determined by PCR-based radiation hybrid mapping, and the gene was located to centromere region of chromosome 11 between markers NIB1900 and D11S1350. Taken together, the VTS approach should provide a novel cDNA cloning strategy for isolating unidentified genes, which are not found even in EST databases but are detectable computationally.

Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells.

The aim of this study was to investigate the expression of the muscarinic acetylcholine receptor (mAchR) subtypes mRNA in primary culture of human prostate stromal cells using the reverse transcription polymerase chain reaction (RT-PCR), RNA blotting and in situ hybridization (ISH). Using an explant method, we obtained a primary culture of prostate stromal cells from three patients with benign prostatic hypertrophy. Total RNA was extracted using the acid guanidinium method for cDNA synthesis. First-strand cDNA was then used for PCR with primers designed to amplify the fragments of each mAchR subtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands were detected; in particular m2 transcripts was strongly detected in the stromal cell culture. Each of the PCR products were subcloned into the pGEM-T plasmid vector, sequenced and random primer labeled using 32P. Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcript. However, m3 and m4 probes did not hybridize. Using in situ hybridization (ISH), m2 receptor mRNA signals were detected in several smooth muscle cells. The staining was predominantly localized to the perinuclear cytoplasm. The m3 and m4 probes did not hybridize. These results suggested that m2 receptor subtype plays a role in smooth muscle activity of the human prostate.

Comparative nucleotide sequence analyses of the entire genomes of B95a cell-isolated and vero cell-isolated measles viruses from the same patient.

Experimental infection of monkeys with the IC-B strain of measles virus (MV), which was isolated in marmoset B lymphoblastoid B95a cells from an acute measles patient, caused clinical signs typical for measles, while infection by the IC-V strain isolated in African green monkey kidney Vero cells from the same patient did not cause any clinical signs in infected monkeys. The IC-B strain replicated only in B95a cells, whereas the IC-V strain replicated in both B95a and Vero cells (3,6). To clarify which gene or mutation(s) was responsible for the difference in these phenotypes, the nucleotide sequences of the entire genomes of the IC-B and IC-V strains were determined. Comparative nucleotide sequence analyses revealed only two nucleotide differences, one in the P/V/C gene and the other in the M gene, predicting amino acid differences in the P, V and M proteins and a 19 amino acid deletion in the C protein of the IC-V strain. The truncation in the C protein was confirmed for the IC-V strain by immunoprecipitation using the C protein specific antiserum. No nucleotide difference was found in the envelope H gene. These results indicated that nucleotide difference(s) in the P/V/C or/and M gene, and not H gene, was responsible for the different cell tropism and pathogenicity of MV in this case.

Recovery of pathogenic measles virus from cloned cDNA.

Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700-705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773-5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.

Computational and experimental analysis identifies many novel human genes.

Because of advances in automation, human genomic sequences are being deposited in public databases at a dramatic rate. However, the process of detecting genes in these sequences is still something of an art. Here we describe the implementation and testing of a relatively straightforward computational approach, the Virtual Transcribed Sequence project, which analyzes their gene content using the gene prediction program GENSCAN (GENSCAN 1.0 1,2) in combination with similarity-based methods. This approach identifies many novel human genes not found even in EST databases.

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.

Structural analysis of Arabidopsis thaliana chromosome 5. IX. Sequence features of the regions of 1,011,550 bp covered by seventeen P1 and TAC clones.

In this series of projects sequencing the entire genome of Arabidopsis thaliana chromosome 5, non-redundant P1 and TAC clones have been sequenced according to the fine physical map, and as of May 7, 1999, the sequences of 16.2 Mb representing approximately 60% of chromosome 5 have been accumulated and released at our web site. In parallel, structural features of the sequenced regions have been analyzed by applying a variety of computer programs, and to date we have predicted a total of 2380 potential protein-coding genes in the 10,154,580 bp regions, which are covered by 142 P1 and TAC clones. In this paper, we newly analyzed the structural features of the 1,011,550 bp regions covered by additional 17 P1 and TAC clones, and predicted 298 protein-coding genes. The average density of the genes identified was 1 gene per 3394 bp. Introns were observed in 67% of the genes, and the average number per gene and the average length of the introns were 3.2 and 159 bp, respectively. The gene density became higher than the value estimated in the previously analyzed regions (1 gene per 4,267 bp), as the data in this paper were compiled based on a new standard of gene assignment including the computer-predicted hypothetical genes. The regions also contained 8 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available on the database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.

Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

To extend our cDNA project for accumulating basic information on unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human adult and fetal brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA1019 to KIAA1118. The sequencing of these clones revealed that the average size of the inserts and corresponding open reading frames were 5.0 kb and 2.6 kb (880 amino acid residues), respectively. Database search of the predicted amino acid sequences classified 58 predicted gene products into the five functional categories, such as cell signaling/communication, cell structure/motility, nucleic acid management, protein management and cell division. It was also found that, for 34 gene products, homologues were detected in the databases, which were similar in sequence through almost the entire regions. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.