Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer.

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common ß subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

Immunohistochemical investigation of mid-dermal elastolysis with a history of erythema.

Elastic fibers are essential extracellular matrix macromolecules comprising an elastin core surrounded by fibrillin-rich microfibrils. Fibulin-5, a microfibril, has been identified as one of the secreted extracellular matrix proteins that shows function as a scaffold for elastic fibers. However, the distribution of fibulin-5 in the skin is not clear. We report a case of a 43-year-old woman with erythema and subsequent wrinkling that met the clinical and histological criteria for mid-dermal elastolysis. We investigate the mechanism by which this disease occurs. The distribution of elastin, CD68, matrix metalloproteinase (MMP)-9, and fibulin-5 was examined immunohistochemically from both erythematous and wrinkled skin. There were numerous CD68 and MMP-9-producing histiocytes and giant cells in the erythematous lesions. Faint fibrillar staining of fibulin-5 was found in the deep dermis. In the wrinkled skin, there were few CD68 histiocytes or giant cells. Elastin immunoreactivity disappeared from the mid-dermis. Fibulin-5 colocalized in the lower dermis, shorter than in the erythema. Mid-dermal elastolysis may be initiated by MMP-9 produced by histiocytes and giant cells through its degradation of elastic fibers. In the lower dermis of the wrinkled skin, the fragmented expression of fibulin-5 was associated with the incomplete reproduction of the elastic fibers.

Vertebrate TBP-like protein (TLP/TRF2/TLF) stimulates TATA-less terminal deoxynucleotidyl transferase promoters in a transient reporter assay, and TFIIA-binding capacity of TLP is required for this function.

The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that artificially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATA-less promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP significantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind specifically to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.

Dioxin induces a novel nuclear factor, DIF-3, that is implicated in spermatogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF-3 (dioxin inducible factor-3), by cDNA representational difference analysis. DIF-3 protein is a nuclear factor and possesses a zinc-finger motif at its N-terminus. High DIF-3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF-3 protein was detected during spermatogenesis. Thus, these results suggest that DIF-3 may be a target gene mediating the reproductive toxicity induced by TCDD.

Promoter structure and gene expression of the mouse inositol 1,4,5-trisphosphate receptor type 3 gene.

Inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) is a ubiquitously expressed IP(3)R gene in the IP(3)R gene family. We identified an upstream region of the mouse IP(3)R3 genomic DNA. Transcription start points for the IP(3)R3 gene were found to be located mainly at four sites between nucleotide position -325 and -285 relative to the first ATG codon. The major start point was mapped around -325. Transcription promotion ability was detected between -325 and -285 in an IP(3)R3 proximal promoter sequence. The promoter had no TATA-box but was highly GC-rich and contained two putative Sp1-binding sites. There was no sequence similarity between promoter regions of IP(3)R3 and IP(3)R2, another ubiquitous gene, except for GC-boxes. By using a series of 5'-truncation versions and a transient luciferase assay, we detected multiple common and cell-type-dependent regulatory regions within the distal promoter sequence downstream from -4.0 kb that function positively or negatively. The IP(3)R3 gene was highly transcribed in the kidney, spleen, heart, and skeletal muscle, and this tissue distribution pattern was nearly complementary to that of IP(3)R2. We found that IP(3)R3 gene expression was repressed in retinoic acid-treated and neural differentiated P19 mouse embryonic carcinoma cells.

Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.

TBP-like protein (TLP/TLF/TRF2) artificially recruited to a promoter stimulates basal transcription in vivo.

Metazoan genomes generally contain one TBP-related gene designated as TBP-like protein (TLP/TLF/TRF2). Although TLP is thought to work for transcriptional regulation, its natural function has not been clearly demonstrated. Here we describe the stimulation of transcription from TATA-containing and TATA-less class II promoters by artificially recruited mammalian TLP. TLP fused with Gal4 DNA-binding domain stimulated transcription when it was recruited at a proximal promoter. Compared to TBP, stimulation by TLP was less TATA-dependent. Slight truncation from each terminus of TLP destroyed this function drastically. Amino acid substitutions of TLP whose corresponding residues in TBP are crucial for its function resulted in the loss of function. Consequently, Gal4-fused TLP was demonstrated to exhibit ability of transcription activation irrespective of the type of promoter, the mechanism of which was thought to be similar to that of artificially recruited TBP. TLP is presumably able to behave as a transcriptional activator in cells.

Enzymatic synthesis of modified DNA by PCR.

We synthesized various 5'-triphosphates of C5-substituted 2'-deoxyuridine derivatives bearing methylene linker at C5-alpha position. We examined whether the C5-substituted 2'-deoxyuridine 5'-triphosphates (dUTP) can work as a substrate for the modified DNA synthesis by PCR. We found that only KOD dash DNA polymerase, a thermostable DNA polymerase from extremely thermophilic archaeum, accepted the modified substrates in place of TTP for PCR forming the corresponding modified DNAs. On the other hand, no other DNA polymerase could accept these TTP analogues.

[A case of successful management of nonresectable advanced cholangiocellular carcinoma by intermittent hepatic arterial infusion at home].

An 82-year-old female was referred to our hospital because a 16 x 8 cm tumor detected in her liver by abdominal ultrasonography (echo, hereafter) and CT. The patient was diagnosed as having highly advanced cholangiocellular carcinoma (CCC) by abdominal angiography. Since excision of the tumor was impossible due to the patient's age, a reservoir was indwelled for intra-arterial injection into the liver. Continuous injection of 1,000 mg 5-FU over 24 hours was performed every 2 weeks using a portable disposable pump 70 times. The tumor has been markedly reduced since the start of chemotherapy, with a reduction rate (PR) of 98% over the 3 years until the present. Since the frequency of administration was low, only twice a month, the patient had few side effects despite her old age, and injections could be performed in the outpatient department. Usually, the prognosis for CCC is poor. However, the patient has maintained a good QOL with the periodic intra-arterial injection of the carcinostatic into the liver, and this treatment has had a strong antitumor effect. This chemotherapy is thus considered useful for CCC which can not be resected.

Differential Expression of Cyclins B1 and B2 during Medaka (Oryzias latipes) Spermatogenesis.

The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.

[Five cases of cerebral and/or cerebellar embolism after insertion of a heparin-coated catheter from the left thoracoacromial artery].

We reported five cases of cerebral and/or cerebellar embolism after insertion of a heparin-coated catheter from the left thoracoacromial artery for multiple liver metastases. They were one patient with multiple liver metastases from the angiosarcoma of the scalp, and 4 others with gastrointestinal cancer liver metastases. The first case suffered a cerebeller embolism just after removal of a catheter that had been obstructed. In this case, it is possible that the thrombus quickly migrated into a cranial vessel from around the catheter. In the others with patent catheters, the cerebral embolisms occurred more than a month after insertion of the catheters. In the latter cases, it is thought that embolisms did not occur because of catheter insertion maneuver. However, a thrombus that grew around the catheter migrated into the left common carotid artery or the left vertebral artery. The anti-coagulation therapy should be considered for prophylactic treatment.

Analysis of the chicken TBP-like protein(tlp) gene: evidence for a striking conservation of vertebrate TLPs and for a close relationship between vertebrate tbp and tlp genes.

TLP (TBP-like protein), which is a new protein dis-covered by us, has a structure similar to that of the C-terminal conserved domain (CCD) of TBP, although its function has not yet been elucidated. We isolated cDNA and genomic DNA that encode chicken TLP (cTLP) and determined their structures. The predicted amino acid sequence of cTLP was 98 and 91% identical to that of its mammalian and Xenopus counterparts, respectively, and its translation product was ubiquitously observed in chicken tissues. FISH detection showed that chicken tlp and tbp genes were mapped at 3q2.6-2.8 and 3q2.4-2.6 of the same chromosome, respectively. Genome analysis revealed that the chicken tlp gene was spliced with five introns. Interestingly, the vertebrate tbp genes were also found to be split by five introns when we focused on the CCDs, and their splicing points were similar to those of tlp. On the contrary, another TBP-resembling gene of Drosophila, trf1, is split by only one intron, as is the Drosophila 's tbp gene. These results support our earlier assumption that vertebrate TLPs did not directly descend from Drosophila TRF1. On the basis of these results together with phylogenetical exam-ination, we speculate that tlp diverged from an ancestral tbp gene through a process of gene duplication and point mutations.

Isolation of cDNA, chromosome mapping, and expression of the human TBP-like protein.

TBP is an essential factor for eukaryotic transcription. In this study, we identified a human cDNA encoding 21-kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of human TBP with 39% identity and 76% similarity. FISH determined that human tlp gene was located at chromosome 6 region q22.1-22.3. Northern blot analysis demonstrated that TLP mRNAs were expressed in various human tissues ubiquitously. We found that the TLP proteins exist in multiple mammalian cells and chicken cells. Although the Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor, expression of TLP was nearly constant throughout the neural differentiation of P19 cells. Unlike TRF, TLP did not bind to the TATA-box nor direct transcription initiation in vitro. Similarity between TRF and TLP was considerably lower (35 in alignment score) than that between Drosophila TBP and human TBP (88 in alignment score). Multiple amino acids critical for the TBP function were deleted or substituted in TLP. We suggest that TLP is not a bona fide vertebrate counterpart nor a direct descendant of TRF.

Identification of a mouse TBP-like protein (TLP) distantly related to the drosophila TBP-related factor.

TATA-binding protein (TBP) is an essential factor for eukaryotic transcription. In this study, we demonstrated a mouse cDNA encoding a 21 kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of mouse TBP with 39% identity and 76% similarity. Northern blot analysis demonstrated that TLP mRNAs were expressed in various mammalian tissues ubiquitously and that their distribution pattern was analogous to that of TBP. By using anti-TLP antibody, we demonstrated the existence of TLP proteins in various mammalian cells and tissues. The Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor that binds to the TATA-box and activates transcription. TLP did not bind to the TATA-box nor direct transcription initiation. Multiple amino acids critical for TBP function were deleted or substituted in TLP, while amino acids in Drosophila TRF much resembled those in TBP. Similarity between Drosophila TRF and mouse TLP was considerably lower (alignment score 35) than that between Drosophila TBP and mouse TBP (alignment score 88). Identity of nucleotide sequences between mouse and putative human TLPs (94%) was higher than that between TBPs (91%) in these two animals. Expression of TLP was nearly constant throughout the P19 differentiation process. Accordingly, we suggest that, even if higher eukaryotes generally contain multiple tbp -related genes, TLP is not a bona fide mammalian counterpart of Drosophila TRF.

[Angiographic diagnosis and management of bleeding from the digestive organs].

Gastrointestinal bleeding is recently seen less often by the angiographer. This is mainly due to advances in endoscopy, and nuclear medicine. When patients with gastrointestinal bleeding are referred, endoscopic diagnosis and therapy should be performed at first. However, when it is impossible to diagnose or to control the bleeding, angiography must be considered as soon as possible. Intra-abdominal bleeding should be diagnosed by angiography at first. In both cases, embolization is generally safe and effective depending on the advance of occlusive agents.

Transcription initiation sites and promoter structure of the mouse type 2 inositol 1,4,5-trisphosphate receptor gene.

Transcription initiation sites and the promoter sequence of the ubiquitously expressed mouse type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) gene were determined. In contrast to the nervous system-enriched IP3R1, the IP3R2 gene had multiple (seven major) transcription initiation sites located 334 to 269 bp upstream from the first ATG codon. Transient luciferase assay revealed promoter activity of the IP3R2 sequence upstream from the transcription initiation sites. The IP3R2 promoter was GC-rich and had no conventional TATA box, but had a GC box in the proximal promoter. Multiple transcription start sites were flanked by CpG islands, and various cis elements were located in the promoter. These structural features are considered to be responsible for a profile of IP3R2 gene expression.

Spermatid-specific overexpression of the TATA-binding protein gene involves recruitment of two potent testis-specific promoters.

The gene encoding the TATA-binding protein, TBP, is highly overexpressed during the haploid stages of spermatogenesis in rodents. RNase protection analyses for mRNAs containing the previously identified first, second, and eighth exons suggested that most TBP mRNAs in testis did not initiate at the first exon used in somatic cells (here designated exon 1C). Using a sensitive ligation-mediated cDNA amplification method, 5' end variants of TBP mRNA were identified, and the corresponding cDNAs were cloned from liver and testis. In liver, a single promoter/first exon is used to generate a steady-state level of roughly five molecules of TBP mRNA per diploid cell equivalent. In testis, we detect modest up-regulation of the somatic promoter and recruitment of at least five other promoters. Three of the alternative promoter/first exons, including 1C and two of the testis-specific promoter/first exons, 1D and 1E, contribute roughly equivalent amounts of mRNA which, in sum, account for greater than 90% of all TBP mRNA in testis. As a result, round spermatids contain an estimated 1000 TBP mRNA molecules per haploid cell. Testis TBP mRNA also exhibits several low abundance 5' end splicing variants; however, all detected TBP mRNA leader sequences splice onto the common exon 2 and are expected to initiate translation at the same site within exon 2. The precise locations of the three major initiation exons are mapped on the gene. The identification of the strong testis-specific promoter/first exons will be important for understanding spermatid-specific tbp gene regulation.

Promoter structure of the mouse TATA-binding protein (TBP) gene.

5'-RACE and genomic cloning were used to determine that the mouse TBP (mTBP) gene consists of one 5'-terminal non-coding exon followed by seven protein-coding exons. The region upstream of the first exon lacks a TATA-box. Hence, as with the case of other genes carrying TATA-less promoter, transcription starts from a cluster of sites which are located at the restricted region of mTBP gene. Interestingly, sequences of this region are well conserved between human and mouse TBP genes, suggesting that both mouse and human TBP genes drive mRNA synthesis in a similar way, and the sequence homology between two genes was used to assign a putative start point for the human TBP gene. Mouse, human, and Trimersurus gramineus (green habu snake) TBP genes share two GC-rich regions in their promoter regions. Thus, it is probable that diverse species of vertebrates commonly use TATA-less promoter bearing GC-rich regions to direct ubiquitous TBP expression.

[Hemodynamic effects of amrinone on patients with moderate and severe cardiac dysfunction after open heart surgery].

Hemodynamic effects of amrinone and the changes in pulmonary shunt were studied in patients for open heart surgery. Eighteen patients were allocated into two groups (group L, H) on the first day of surgery. Group L consisted of 10 patients who received moderate-dose catecholamine (DOA + DOB < 12 micrograms.kg-1.min-1) and group H consisted of 8 patients who needed high-dose catecholamine (DOA + DOB > or = 12 micrograms.kg-1.min-1) to maintain normal hemodynamics. After baseline measurement, amrinone infusion was started and the dose was increased by every 2 hours (5, 10, 20 micrograms.kg-1.min-1). At dose of 20 micrograms.kg-1.min-1, cardiac index increased significantly in group L but not in group H. SVRI and PVRI decreased by dose related fashion in both groups. Systemic arterial pressure was unaltered in group L while it decreased significantly in group H. PaO2 decreased and pulmonary shunt increased in both groups. These results suggest that inotropic response of amrinone depends on the basal level of myocardial contractility although vasodilative property and the effect on the pulmonary shunt are almost similar in both groups.

[Malignant fibrous histiocytoma of the spermatic cord: a case report].

A case of malignant fibrous histiocytoma of the spermatic cord is reported. A 44-year-old man was admitted because of a painless, gradually enlarging mass in the left scrotum. Local tumor excision and subsequent radical inguinal orchidectomy was performed. The histological diagnosis was malignant fibrous histiocytoma. There were no signs of recurrence or metastasis 8 months after the operation. Malignant fibrous histiocytoma in the spermatic cord is rare. We reviewed 34 previously reported cases. The rate of local recurrence and distant metastasis are 34.5% and 17.2%, respectively.