PARVB overexpression increases cell migration capability and defines high risk for endophytic growth and metastasis in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is highly diverse, even in its early stages. This cancer is classified into three subtypes (superficial, exophytic, and endophytic) based on macroscopic appearance. Of these subtypes, the endophytic tumours have the worst prognosis because of their invasiveness and higher frequency of metastasis. METHODS: To understand the molecular mechanism underlying the endophytic subtype and to identify biomarkers, we performed a comprehensive gene expression microarray analysis of clinical biopsy samples and also confirmed the clinical relevance of differential gene expression. RESULTS: Expression of the parvin-beta (PARVB) gene and its encoded protein was significantly upregulated in endophytic-type TSCC. PARVB is known to play a critical role in actin reorganization and focal adhesions. Knockdown of PARVB expression in vitro caused apparent decreases in cell migration and wound healing, implying that PARVB has a crucial role in cell motility. Moreover, metastasis-free survival was significantly lower in patients with higher tumour expression of PARVB. CONCLUSIONS: These findings suggest that PARVB overexpression is a candidate biomarker for endophytic tumours and metastasis. This protein may be a clinically useful target for adjuvant TSCC therapy.

GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

Development of a telediagnosis endoscopy system over secure internet.

OBJECTIVES: We developed a new telediagnosis system to securely transmit high-quality endoscopic moving images over the Internet in real time. This system would enable collaboration between physicians seeking advice from endoscopists separated by long distances, to facilitate diagnosis. METHODS: We adapted a new type of digital video streaming system (DVTS) to our teleendoscopic diagnosis system. To investigate its feasibility, we conducted a two-step experiment. A basic experiment was first conducted to transmit endoscopic video images between hospitals using a plain DVTS. After investigating the practical usability, we incorporated a secure and reliable communication function into the system, by equipping DVTS with "TCP2", a new security technology that establishes secure communication in the transport layer. The second experiment involved international transmission of teleendoscopic image between Hawaii and Japan using the improved system. RESULTS: In both the experiments, no serious transmission delay was observed to disturb physicians' communications and, after subjective evaluation by endoscopists, the diagnostic qualities of the images were found to be adequate. Moreover, the second experiment showed that "TCP2-equipped DVTS" successfully executed high-quality secure image transmission over a long distance network. CONCLUSIONS: We conclude that DVTS technology would be promising for teleendoscopic diagnosis. It was also shown that a high quality, secure teleendoscopic diagnosis system can be developed by equipping DVTS with TCP2.

Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells.

Laminin (LN)-5, a heterotrimer of alpha3, beta3, and gamma2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN gamma2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN gamma2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN gamma2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN alpha3 and beta3 chains, suggesting that the LN gamma2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN gamma2 chain without any detectable signal for the LN alpha3 or beta3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN gamma2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN gamma2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets.

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.

Development of the real-time control (RTC) system for Tokyo sewage system.

Tokyo Metropolitan government has decided to make the maximum possible use of the existing facilities while ensuring safety against inundation and to promote measures also from a software approach by introducing a system capable of minimizing combined sewer overflow, the real-time control system (RTC). A pilot RTC system was installed in August 2002 for the Shinozaki Pumping Station. The RTC system monitors the precipitation volume and the water level in the pipe. Simulations were carried out on the basis of these data. From the results, it was found that with the use of the RTC it is possible to reduce CSO by roughly 50% for small rainfalls with a total precipitation level of 20 mm or less by strong rainwater in the pipe routes at the beginning of the rain. It has also been shown that CSO can be reduced by about 80% through the use of rainfall forecasting.

Assignment of human membrane-type matrix metalloproteinase-2 (MT2-MMP) gene to 16q12 by FISH and PCR-based human/rodent cell hybrid mapping panel analysis.

A new group of matrix metalloproteinase with a potential membrane domain was reported as membrane-type matrix metalloproteinases (MT-MMPs), and the gene coding for one of them, MT2-MMP was recently cloned from a human lung cDNA library. To predict its physiological functions by the relation to the genetic disorders mapped on the chromosomes, the chromosomal location of the human MT2-MMP gene was examined by fluorescence in situ hybridization (FISH) and PCR-based analysis with human/rodent hybrid cell mapping panels, and it was assigned to 16q12.

Characterization and transcriptional regulation of rat glutamine tRNA-encoding genes: repression of tRNA(UUGGln) synthesis.

In rat liver, the amount of tRNA(UUGGln) (RG-2) was found to be approx. 20% of that of tRNA(CUGGln) (RG-1). The independent RG-1 and RG-2 genes were isolated from a rat genomic library together with four RG-related genes containing two to five alterations in their coding regions. Sequence analysis demonstrated that there was no difference between the internal promoter sequences of RG-1 and RG-2. However, interestingly, the transcriptional activity of RG-1 was approximately four-times higher than that of RG-2 in an in vitro transcription reaction. Replacement of the 5'-flanking sequence of RG-2 by the corresponding sequence of RG-1 or by a plasmid DNA sequence caused activation of RG-2 transcription. Gel retardation assay demonstrated that the 5'-flanking region of RG-2 contained a unique sequence specifically recognized by a nuclear protein. Taken together, these results strongly suggest that the transcriptional activity of RG-2 might be negatively regulated by the binding of a nuclear protein at a specific site in the 5'-flanking region of the gene.

Processing of hepatitis C viral polyprotein in Escherichia coli.

Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2. Cpro-1 and Cpro-2 appear to process the precursor polyprotein from which they originate. Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusion proteins. The N- and C-terminal ends of the HCV polypeptides were fused with the E. coli maltose-binding protein (MBP) and E. coli dihydrofolate reductase (DHFR), respectively. The proteinase activities cleaved the fusion polypeptides by the same processing pathway used in eukaryotic protein production systems. The N-terminal amino acid (aa) sequences of the processed fusion proteins were determined. A comparison of those N-terminal sequences with the aa sequence of the HCV precursor polyprotein showed that the N-terminal and C-terminal cleavage sites of p70(NS3), one of the HCV nonstructural (NS) proteins, were the same as those identified in other processing studies: cleavages were estimated to be between aa 1026 and 1027 and between aa 1657 and 1658 of the HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively. Cpro-1 and Cpro-2 both functioned in E. coli and possessed authentic characteristic features.

Expression of trypsin in vascular endothelial cells.

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen-2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell-derived trypsin in tumor angiogenesis and abnormal blood coagulation.

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Inherited primary hypothyroidism with thyrotrophin resistance in Japanese cats.

Mutant cats were developed with non-goitrous primary hypothyroidism. They were clinically characterized by severely retarded growth, mild anaemia and high mortality in the young. They responded markedly to thyroid hormone replacement. Thyroid glands in the mutants were normal in position but slightly reduced in size. Laboratory studies revealed low serum concentrations of thyroxine (T4) and tri-iodothyronine (T3), and increased serum concentrations of TSH. Administration of TRH induced no further increase in TSH. Administration of exogenous TSH after suppression of endogenous TSH by T3 did not increase the serum concentration of T4 in the mutants, in sharp contrast with the threefold increase in serum T4 observed in the normal litter-mates. These findings suggest that the underlying pathogenesis of this disorder is unresponsive to TSH. Moreover, we found that the mutants were transmitted in an autosomal recessive manner.

Association between dopamine D4 receptor (D4DR) exon III polymorphism and novelty seeking in Japanese subjects.

This study was designed to assess the association between novelty seeking and D4DR gene polymorphism in the Japanese population. The 48 bp repeat polymorphism in the third exon of the dopamine D4 receptor gene of 153 normal female students was correlated with personality feature results from the Japanese version of Cloninger's Temperament and Character Inventory. The Novelty Seeking subscale of Exploratory Excitability had a significant association with long alleles of the polymorphic exon III repeat sequence of D4DR. Our results suggest that there is an association between long alleles of the polymorphic exon III repeat sequence of D4DR and the personality traits of the Novelty Seeking subscale of Exploratory Excitability, regardless of racial differences in the frequencies of D4DR exon III repeat polymorphism.

Structure of cDNA coding for rat platelet phospholipase A2.

Three cDNA clones coding for rat platelet phospholipase A2 and a homologous protein were isolated from a rat megakaryocyte cDNA library and sequenced. One (prPLA2-1) carries a 708 nucleotide long insert. The others (prPLA2-2 and -3) differ from clone 1 in three nucleotides and have a 748 nucleotide long insert. All contain a single open reading frame which encodes a 146 amino acid long polypeptide. Based on the deduced amino acid sequence, we concluded that prPLA2-1 encodes rat platelet phospholipase A2. prPLA2-2 and -3 most probably encode a protein homologous to phospholipase A2 with two amino acid replacements. A typical signal peptide sequence (21 amino acid long), located at the NH2 termini of the deduced structure, was immediately followed by a polypeptide which corresponds to the mature enzyme, suggesting that the rat platelet enzyme is not expressed as a proenzyme form. Northern blot analysis showed a single transcript, which is 900 to 1,100 nucleotides long, in the poly(A)+RNA fractions of rat megakaryocytes, bone marrow cells, peritoneal cells of caseinate-treated rats, and spleen cells.

Differential expression of laminin-5/ladsin subunits in human tissues and cancer cell lines and their induction by tumor promoter and growth factors.

We previously reported a new laminin variant containing laminin gamma 2 (or B2t) chain, ladsin, which exerted prominent cell-scattering, cell-adhesion, and cell-migration activities. In the present study, this laminin was further characterized, and gene expression of its three subunits in various human tissues and cancer cell lines was examined by Northern blotting. cDNA cloning of the largest subunit of ladsin and partial amino acid sequencing of its beta (or B1) subunit revealed that ladsin was identical to laminin-5 (kalinin/epiligrin/ nicein). Among various human tissues, placenta, lung, and fetal kidney expressed high levels of mRNAs for the three subunits of laminin-5 (laminin alpha 3EPA, beta 3, and gamma 2 chains). Most gastric and squamous carcinoma cell lines constitutively expressed all of the three subunit mRNAs, while other types of carcinoma cell lines expressed one or two of them. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) strongly enhanced the gene expression of the three subunits, increasing 2 to 8-fold the secretion of laminin-5 from carcinoma cells into culture medium. However, TPA treatment did not increase the secretion of laminin beta 1 chain, a subunit of laminins-1, -3, and -6. The unique properties and inducibility by TPA and EGF of laminin-5 suggest that it is associated with growth and migration of cancer cells.

Lysophospholipase L1 from Escherichia coli K-12 overproducer.

After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.

Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli.

The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.

The DNA sequence encoding pldA gene, the structural gene for detergent-resistant phospholipase A of E. coli.

The nucleotide sequence of the pldA gene of Escherichia coli K-12, which codes for detergent-resistant phospholipase A (DR-phospholipase A), located in the outer membrane, was determined and the amino acid sequence of DR-phospholipase A was deduced. DR-phospholipase A contains 269 amino acids, resulting in a protein with a molecular weight of 30,809. It does not contain any cysteine residues and seems to be synthesized first as a precursor with a typical signal peptide composed of 20 amino acids. The NH2-terminus of the mature protein is glutamine, a polar amino acid, while other outer membrane proteins so far determined have a nonpolar amino acid there. The hydropathy profile of the deduced amino acid sequence of DR-phospholipase A was studied. Most of the region was rather hydrophilic and there were no stretches of hydrophobic amino acids. Computer analysis showed that there are no homologies between DR-phospholipase A and other extracellular phospholipases whose amino acid sequences are known. The candidates for the promoter region of the pldA gene, the 5'-flanking region, have a significantly high AT content, while the AT content of the coding region is about the same as the average AT content of the E. coli chromosome. A typical rho-independent transcription termination site is also present at the 3'-flanking region. This is the first example of the primary structure of a membrane-bound phospholipase.

Role of tissue inhibitor of metalloproteinases-2 (TIMP-2) in regulation of pro-gelatinase A activation catalyzed by membrane-type matrix metalloproteinase-1 (MT1-MMP) in human cancer cells.

To clarify the regulatory mechanism of pro-gelatinase A (proGelA) activation at a cellular level, expression of gelatinase A (GelA), three MT-MMPs, and TIMP-2 was examined with 11 human cancer cell lines cultured in the presence and absence of stimulants. MT1-MMP mRNA was expressed in 8 cell lines, while MT2-MMP and MT3-MMP mRNAs were expressed in fewer cell lines. The cells with high proGelA activation strongly expressed MT1-MMP mRNA but not MT2-MMP and MT3-MMP mRNAs, suggesting that MT1-MMP was responsible for the proGelA activation in the cancer cells. Treatments with concanavalin A (Con A) and a phorbor ester (TPA) enhanced the MT1-MMP expression, but only Con A stimulated the proGelA activation in many cell lines. In HT1080 fibrosarcoma cells, however, TPA also stimulated the activation. The level of TIMP-2 secreted into culture medium inversely correlated with proGelA activation. For example, 2 squamous cell carcinoma lines (HSC-3 and HSC-4) and 3 HT1080 clones, which efficiently activated proGelA, secreted little TIMP-2 into medium, whereas other cell lines and other HT1080 clones, which hardly activated proGelA, secreted TIMP-2 at high levels. When HSC-3 cells were incubated with TIMP-2 protein or transfected with TIMP-2 cDNA, the proGelA activation was strongly inhibited. These results indicated that extracellular TIMP-2 was an important negative regulator of proGelA activation. However, the level of extracellular TIMP-2 was not consistent with that of TIMP-2 mRNA in some cell lines. Other experimental results suggested that TIMP-2 might be rapidly metabolized after binding to MT1-MMP, and Con A treatment might stabilize the complex of TIMP-2 and MT1-MMP on cell membranes.

Detection and subcellular localization of rabbit platelet phospholipase A2 which preferentially hydrolyzes an arachidonoyl residue.

Like rat platelets, rabbit platelets contain a secretory 14-kDa group II phospholipase A2 [Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M., Kim, D.K., Kondo, E., Tomita, M., & Inoue, K. (1989) J. Biochem. 105, 520-525]. The present study was undertaken to determine whether or not, in addition to that of the 14-kDa group II enzyme, rabbit platelets exhibit another phospholipase A2 activity. A rabbit platelet soluble fraction was prepared by sonication and centrifugation. When this soluble fraction was subjected to heparin-Sepharose column chromatography, phospholipase A2 activity was detected in both heparin-binding and heparin-non-binding fractions. The activity detected in the heparin-binding fraction appeared to belong to the secretory 14-kDa phospholipase A2, because it bound to anti-human 14-kDa group II phospholipase A2 monoclonal antibody. The activity found in the heparin-non-binding fraction did not appreciably react with the same antibody. When platelets were gently disrupted by the nitrogen cavitation method, the heparin-non-binding activity was mainly recovered in the platelet cytosolic fraction. The heparin-non-binding phospholipase A2 hydrolyzed a phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than one with a linoleoyl residue. The biochemical features of the activity observed in the heparin-non-binding fraction generally resembled those of human platelet soluble phospholipase A2 [Kim, D.K., Kudo, I., & Inoue, K. (1988) J. Biochem. 104, 492-494].(ABSTRACT TRUNCATED AT 250 WORDS)