Endoscopic intermuscular dissection (EID) for removing early rectal cancers and benign fibrotic rectal lesions.

In the current era of screening colonoscopy and increasing incidence of early rectal cancer, interventional endoscopy moves toward resections in deeper planes than the submucosal layer. Several reports support the use of endoscopic intermuscular dissection (EID) instead of endoscopic submucosal dissection (ESD) for the removal of deeply invasive rectal submucosal cancers. The resection plane into the intermuscular space, the space between the longitudinal (external) and circular (internal) muscle layer, allows radical removal of rectal invasive submucosal cancers. Furthermore, the technique offers the potential for dissection of scarred and severe fibrotic lesions in the rectum by cutting deeper and performing a partial myectomy avoiding the narrow submucosal space. We present 23 cases of EIDs both for deeply invasive rectal cancers and benign rectal lesions. This is the first report in the literature of EID resections for malignant and benign disease, including cases of severely fibrotic rectal lesions.

Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein.

The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline-rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis-regulatory sequences required for the L1 layer-specific expression, a series of 5' deletions of the PDF1 promoter were fused to the beta-glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1-specific expression of PDF1 is confined within a 260-bp fragment upstream of the transcription start site. We identified an 8-bp motif in this region that is conserved between promoter regions of all the L1-specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1-specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 l1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1-specific expression.

Identification and characterization of two genes encoding the eukaryotic initiation factor 4A in rice.

A cDNA encoding eukaryotic translation initiation factor 4A (eIF4A) was isolated from a cDNA library of rice (Oryza sativa L.). Based on this DNA sequence, a 414-amino acid protein exhibiting 67, 64 and 59% homology to the mouse, Schizosaccharomyces pombe and Saccharomyces cerevisiae eIF4A, respectively, was predicted. The deduced amino acid sequence contains the characteristic motifs shared by the DEAD box supergene family. Another cDNA of rice eIF4A was reported previously. Comparison of the coding sequences of the two rice eIF4As showed 85% homology in the nucleotide sequence and 90% homology in the amino acid sequence. The genomic clones corresponding to the two rice eIF4A cDNAs were also isolated from a genomic library of rice (Oryza sativa L.). It was found that the two genes have common patterns of exon-intron boundaries. Their coding regions are split into four exons, and there is an additional exon in the 5'-non coding region.

Salicylic acid induces the expression of a number of receptor-like kinase genes in Arabidopsis thaliana.

Receptor-like protein kinases (RLKs) are encoded by a divergent multigene family and their functions have been implicated in a wide range of signal transduction pathways. In this study, we examined the effect of salicylic acid (SA) on the expression of RLK genes in Arabidopsis thaliana. RNA gel blot analysis revealed that transcripts of RKC1 and a number of its homologs, whose translation products contain C-X8-C-X2-C motifs in the putative extracellular domain, accumulated to a higher level in response to SA treatment of plants. The chimeric fusion between the RKC1 5'-upstream region and the beta-glucuronidase (GUS) reporter gene reproduced the SA responsiveness in transgenic plants. In addition, some of RLK genes of the leucine-rich repeat (LRR) class and those of the S-domain class were also induced by SA. We found that the upstream regions of these SA-responsive RLK genes contain the TTGAC sequence, which has been suggested to be important for induced expression of many plant defense genes. These results suggest the involvement of a number of RLKs in SA-mediated defense responses.

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase.

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.

Heat-shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T-DNA insertions.

This paper describes expression profiles of the Arabidopsis HSP18.2 heat-shock gene promoter by using three different reporter genes, and the application of this promoter to a method we have developed to drive heat-shock-dependent transcription of plant genome DNA flanked by T-DNA insertions. We show that, irrespective of the location of the T-DNA insertion, an HSP18.2 promoter towards the left border of the T-DNA effectively induces transcription of flanking genome sequences in response to heat shock. If polyadenylated, tagged transcripts can be easily identified by RT-PCR.

An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana.

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.

Isolation and characterization of copia-type retrotransposons in Arabidopsis thaliana.

We isolated two copia-type retrotransposons from Arabidopsis thaliana. We named these elements AtRE1 (Arabidopsis thaliana Retro Element 1) and AtRE2. Nucleotide sequence analysis revealed that both elements have long terminal repeats (LTRs), and that their internal sequences include one large open reading frame that could encode Gag protein, protease, integrase, reverse transcriptase, and RNaseH. The deduced amino acids sequences contain several domains that are conserved among a large family of retrotransposons. The primer binding site for first-strand DNA synthesis and the polypurine tract for second-strand DNA synthesis existed at corresponding positions. A 5bp target site duplication (TSD) sequence was also found in the flanking region of LTRs. Southern hybridization and sequence determination of the flanking region demonstrated that AtREs exist at different loci in the two A. thaliana ecotypes Columbia and Landsberg erecta. Moreover, AtRE2 exists at two loci in Landsberg erecta, in contrast to the existence of only one copy in Columbia. These findings suggest that AtREs were recently transfected via some mediators or that AtREs were transposed after differentiation of the two ecotypes. One cDNA clone derived from the transcripts of AtRE1 was isolated, and the nucleotide sequence showed that this RNA was transcribed in the antisense direction. RT-PCR analysis revealed that AtRE1 was transcribed in both directions. This result suggests that the antisense RNA controls the expression of AtRE1 at the post-transcriptional level.

Identification and characterization of the gene encoding the mitochondrial elongation factor G in rice.

A plant nuclear gene coding for a mitochondrial elongation factor G (mEF-G) was cloned from a cDNA library and genomic library of rice (Oryza sativa L.). This DNA sequence predicts a 757-amino-acid protein exhibiting 79%, 55% and 49% homology to Arabidopsis thaliana, Saccharomyces cerevisiae and rat mEF-G respectively, 53% homology to the elongation factor G in Escherichia coli and 43% homology to soybean chloroplast elongation factor G. The deduced amino acid sequence contains the characteristic motifs shared by all GTP binding proteins. Comparison of the sequence of the genomic clone to that of the cDNA clone revealed that this gene is split nineteen times by introns, although the gene of Arabidopsis is split seventeen times by introns. Some of the introns found in the rice genome are relatively long and they result in a long gene with a size of approximately 15 kb.

Isolation and analysis of cDNA within a 300 kb Arabidopsis thaliana genomic region located around the 100 map unit of chromosome 1.

In order to analyze the organization of genes located at the 100 map unit of chromosome 1, we screened cDNAs hybridized with approximately 300kb contiguous DNA using four P1 clones and one YAC clone. A total of 40 kinds of cDNA were isolated, and their entire sequences were determined. A comparison with the GenBank/EMBL database indicated that three of the cDNAs have been found in Arabidopsis, and that similar sequences to 18 of the cDNAs had been detected in Arabidopsis or other organisms. cDNAs were aligned on a physical map of the contiguous DNA, and the transcriptional direction of each cDNA was determined. This contiguous DNA contains a large direct repeat, which contains five genes. In addition, identical or very similar sequences to two cDNAs are located in a narrow region. Thus, a total of 50 genes were identified, and the gene density was revealed to be approximately one gene every 6kb. In addition, cDNA sequencing revealed the existence of unusual transcripts. A sequence of seven cDNAs seemed to have no significant open reading frames. Furthermore, the existence of antisense RNA and the possibility of alternative splicing were also revealed.

Expression of endoxyloglucan transferase genes in acaulis mutants of Arabidopsis.

A mutant of Arabidopsis with reduced internodal cell length, acaulis5 (acl5), has recently been shown to have reduced transcript levels of a gene for endoxyloglucan transferase, EXGT-A1 (Y. Hanzawa, T. Takahashi, Y. Komeda [1997] Plant J 12: 863-874). In the present study, we cloned genomic fragments of five members of the EXGT gene family, EXGT-A1, EXGT-A3, EXGT-A4, XTR2, and XTR3, and examined their expression in the wild type and in a series of acl mutants. In wild-type plants, the EXGT-A3 gene showed higher expression in lower internodes (internodes between nodes bearing axillary shoots) than in upper and young internodes, in which EXGT-A1 was highly expressed. EXGT-A4 was preferentially expressed in roots and XTR3 in siliques. The XTR2 gene was constitutively expressed. In acl1, acl3, and acl4 mutants, which have a severe defect in leaf expansion as well as in internode elongation, the EXGT-A1 gene showed reduced levels of expression before bolting of plants. In contrast, XTR3 was increased in these mutant seedlings. Reduction of EXGT-A1 expression was also detected after bolting of all acl mutants except acl2, whose growth defect is restricted to lower internodes. These results suggest the involvement of each EXGT in different aspects of organ development.

Cloning and characterization of an L1 layer-specific gene in Arabidopsis thaliana.

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed.

The Arabidopsis ERECTA gene is expressed in the shoot apical meristem and organ primordia.

In Arabidopsis thaliana (L.) Heynh, the mutation in ERECTA is known to confer a compact inflorescence by a reduction in the lengths of internodes and pedicels. We analyzed the expression pattern of this gene during plant development. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with the ERECTA promoter fused to the beta-glucuronidase (GUS) gene, showed that ERECTA was predominantly expressed in the shoot apical meristems and organ primordia. ERECTA expression in the shoot apical meristem was weak early in plant development but increased with the transition from the vegetative to the reproductive growth phase. ERECTA was also strongly expressed in organ primordia and immature organs but weakly in mature organs. Thus, ERECTA was expressed in a cell-specific and developmentally regulated manner. In order to identify the regulatory mechanism responsible for the expression pattern of ERECTA, the cis-acting regions in the ERECTA promoter were defined by study of the expression of the chimeric genes that consist of the 5'- or internal deleted promoter and a GUS reporter gene in transgenic plants. The results showed that the essential cis-regulatory elements governing the spatially and temporally specific expression of ERECTA are located between positions -462 and -228 bp and between positions -228 and -153 bp with respect to the transcriptional initiation site.

Arabidopsis thaliana ECP63 encoding a LEA protein is located in chromosome 4.

DCECP63 is a carrot embryogenic cell protein. To perform its genetic analysis, we isolated an Arabidopsis thaliana (At) ECP63 cDNA. The cDNA encoded a polypeptide of 449 amino acids (aa). Its deduced aa sequence showed extensive similarity with DCECP63, DCDC8 and BPBP8 which are embryonic proteins isolated from carrot and birch, respectively. The aa sequence contained eight well-conserved tyrosine phosphorylation sites and 16 repeats of 11 aa. Southern analysis showed that the AtECP63 gene might belong to a small multigene family. The AtECP63 transcripts accumulated specifically in mature seeds, and exogenous abscisic acid (ABA) induced its expression in immature siliques, but not in vegetative tissues. These results suggested that the AtECP63 gene encoding a putative phosphotyrosine protein belonging to late embryogenesis abundant (LEA) protein in group 3 might be involved in maturation and desiccation tolerance of seeds. Restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) mapping showed that AtECP63 gene was present in the South part of chromosome 4.

ACL5: an Arabidopsis gene required for internodal elongation after flowering.

In rosette plants, the formation of initial flowers is closely linked to the lengthening of internodes (bolting). In order to clarify the molecular basis of bolting, mutants with reduced lengths of internodes were screened. This paper presents the identification and characterization of recessive mutations in ACAULIS5 (ACL5), a gene required for internodal growth in Arabidopsis thaliana. Unlike previously described mutants with reduced size of organs, the acl5 mutant has a severe defect that is restricted to the process of cell elongation after transition to the reproductive stage and shows no phenotype before floral induction. The results of RNA blot hybridizations showed that the acl5 mutation causes a striking reduction in the transcript levels of genes encoding the tonoplast intrinsic protein (gamma-TIP) and the endoxyloglucan transferase (EXGT-A1), both of which have recently been suggested to be important for cell elongation. Furthermore, our morphological study indicates that the mutation also causes proliferative arrest of the apical inflorescence meristem. These results strongly suggest that, during the reproductive phase, the wild-type ACL5 gene product has a critical function not only in the control of elongation growth of organs but also in the continued maintenance of the proliferative activity of flower-producing meristems.

Serum prostate-specific antigen values for the prediction of clinical stage and prognosis in patients with prostate cancer: an analysis of 749 cases.

BACKGROUND: The clinical significance of pretreatment serum prostate-specific antigen (PSA) values was studied to determine the ability to predict clinical stage and prognosis using a relatively large number of patients with prostate cancer. METHODS: Serum PSA values at diagnosis were analyzed from 749 patients with newly-diagnosed prostate cancer and registered in the Tokai Urological Cancer Registry. Correlations between the PSA value, the clinical stage and prognosis of the patients were evaluated. RESULTS: Serum PSA values at each stage of diagnosis showed positivity (> or = 3.6 ng/mL) in 23% (stage A1) to 91.2% (stage D2) of patients, and it was possible to obtain statistical differences between the stages, even between A1 and A2. Based on a cumulative study of PSA distribution, stages greater than A2 could be diagnosed using a cut-off of 7.2 ng/mL, with a 99.2% positive predictive value (PPV), and a 16.2% negative predictive value (NPV). At a PSA level of 10.8 ng/mL, stages greater than B2 could be predicted with a PPV of 95.3% but an NPV of 40.3%. Pretreatment PSA values were a significant prognostic indicator in stage D2 patients using 100 to 150 ng/mL as the cut-off values. These differences were primarily found in the poorly differentiated group, which showed a statistical difference using cut-off PSA values from 75 to 150 ng/mL. CONCLUSIONS: Serum PSA levels from a large number of patients can be used to predict the stage and prognosis of prostate cancer patients.

[Usefulness of 99mTc-DTPA renography and diuretic renography in predicting successful stone discharge following outpatient ESWL in patients with a single ureteral stone].

We analyzed the 99mTc-DTPA renogram with and without diuresis to predict the possibility of stone discharge on the outpatient basis by renogram patterns. Between October, 1993 and December, 1995, 99mTc-DTPA renography was performed in 79 patients with a single ureteral stone. The 99mTc-DTPA renogram pattern was classified into the three types of normal function, obstruction and lower function patterns and the complete stone discharge rate was 93, 63 and 25%, respectively. In addition, diuretic renography using Furosemide was performed in patients with an obstruction pattern and the three renogram patterns of return to the normal curve, a diuretic response and no response were obtained; the complete stone discharge rate was 44, 65.3 and 93%, respectively. From this study, patients with a single ureteral stone with a normal pattern on the regular DTPA renogram and patients with no response pattern on the diuretic renogram, even if in such patients an obstructive pattern was seen on the regular DTPA renogram, seem to be a good candidate for obtaining a high rate of a stone discharge with extracorporeal shock wave lithotripsy (ESWL) treatment in the outpatients basis.

[A case of leiomyoma of the urinary bladder associated with transitional cell carcinoma].

We report a case of leiomyoma of the urinary bladder associated with transitional cell carcinoma. A 60-year-old-male was referred to our hospital because of the complaint of dysuria and for detailed examination of left hydronephrosis. Drip infusion pyelography revealed left uretero-vesico junction stenosis. Flexible cystoscopy revealed benign prostatic hypertrophy and epithelial bladder tumor at the bladder neck and left ureteral orifice. The tumor was histologically diagnosed as TCC (transitional cell carcinoma). M-VAC chemotherapy (methotrexate 30 mg/m2, day 1, 15, 22, vinblastine 3 mg/m2, day 1, 15, 22, adriamycin 30 mg/m2, day 2, cisplatin 70 mg/m2, day 2) was performed as a neoadjuvant chemotherapy. However, since pelvic MRI revealed tumor invasion in to the muscle area, total cystoprostatourethrectomy and ileal conduit were done. Pathological examination of the tumor of left ureteral orifice revealed TCC, G2, INF beta, pT1, ly0, v(-), pN0, PM0. The tumor in the bladder neck was histologically diagnosed as submuscosal type leiomyoma. No cases of leiomyoma of the urinary bladder associated with transitional cell carcinoma have been reported in Japan.