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Biomedical subjects

M Mitsuhashi

Publications and source records attributed to M Mitsuhashi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Inhibitory effect of matrilysin antisense oligonucleotides on human colon cancer cell invasion in vitro.

In colorectal cancer, matrilysin (matrix metalloproteinase-7) is mainly produced by the tumor cells themselves and is thought to play an important role in tumor invasion and metastasis. In the study reported here, we examined the effects of matrilysin antisense phosphorothioate oligonucleotides on both the expression of matrilysin and the invasive potential of the human colon cancer cell line CaR-1 in vitro. To select the most specific and potent oligonucleotide sequence, we performed extensive analyses of the binding specificities of all antisense candidates in the GenBank database by using a computer program we developed. As a result, a 15-mer matrilysin-specific antisense oligonucleotide that hybridizes to the coding region of matrilysin mRNA (AS-1) and a random control oligonucleotide (CL-1) were designed. Reverse transcription-polymerase chain reaction and western blot analysis demonstrated that 10 microM AS-1 suppressed matrilysin expression at both the mRNA level (92%) and protein level (64%). In vitro invasion assays demonstrated that this same concentration of AS-1 inhibited the ability of cells to invade a reconstituted basement membrane by 50% as compared with the ability of untreated cells to do so. On the other hand, CL-1, which had the same length and GC content as AS-1, did not show any inhibitory effect. These results demonstrate that the antisense oligonucleotide AS-1 inhibits matrilysin activities in a sequence-specific manner and suggest that AS-1 has the potential to be used as an anti-metastatic agent in an in vivo experimental model of colon cancer.

Antineoplastic Agents↗
PubMed1998

RNA chip: quality assessment of RNA by microchannel linear gel electrophoresis in injection-molded plastic chips.

Two major components of rRNA (18S and 28S rRNA) were separated by electrophoresis in injection-molded acrylic chips with a microchannel 100 microm in width, 40 microm in depth, and with 1 cm of separation distance. Microchannels were filled with 4 g/L hydroxypropylmethylcellulose as sieving polymer and 5 mg/L ethidium bromide for RNA staining. The fluorescent signals were detected by a fluorescent microscope equipped with a photometer and 590 nm emission filter. The assay is rapid (<3 min), reproducible, RNase-free, and requires only 1-2 microL of sample. The detection limit was approximately 10 mg/L (10 ng/microL), 100-fold lower than that for conventional agarose gel electrophoresis. Because only 0.1 nL of the loaded sample was used for electrophoresis, the detectable peaks of rRNA in the separation were derived from less RNA than in a single cell. Because the quality of RNA is critical for RNA-related diagnostic tests, disposable plastic chips will be useful for quality assessment of RNA.

Animals↗
PubMed1998

Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates.

To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured approximately 40-55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5' end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.

Animals↗
PubMed1997

Rapid cytocidal and cytostatic chemosensitivity test by measuring total amount of mRNA.

Chemosensitivity of vinblastin, cisplatin, and mitomycin C were assessed in four different human cancer cell lines (U937, HL-60, CaR-1, and HepG2) by MTT (3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) assay and the measurement of total cytosolic poly(A) + mRNA. Results of 12 h mRNA assay with 10 x peak plasma concentration (PPC) were significantly correlated with that of standard 3 days MTT assay with 1 x PPC. Furthermore, mRNA assay was changed more significantly than MTT assay under cytostatic condition. Because of its minimum culture requirement (12 h) and broad spectrum (cytocidal and cytostatic), mRNA assay will become a useful tool for chemosensitivity test.

Drug Screening Assays, Antitumor↗
PubMed1997

Strategy for designing specific antisense oligonucleotide sequences.

Antisense compounds, various forms of nucleotides or their analogs, inhibit gene function both in vitro and in vivo. Although antisense compounds have been used extensively not only as a basic research tool but also as therapeutics for various diseases, one of the major problems is the difficulty of obtaining optimal sequences to inhibit specific gene functions. Although the terms "sequence-specificity" or "sequence-nonspecificity" are often used, there is no consensus as to how to define and quantitate such sequence specificity. In this review, we introduced hybridization simulation for designing optimal antisense sequences. Each candidate antisense oligonucleotide is assessed by calculating its hybridization energy against potential hybridization sites within the specified database (including GenBank) using a realistic nearest-neighbor thermodynamic model, taking into account mismatches. The specificity of each oligonucleotide is then quantitated by the number of potential cross-hybridizable genes and their degree of cross-hybridization. Furthermore, if antisense sequences exhibit a high potential for hairpin formation, they are not recommended even if they are highly specific. Therefore, to select antisense sequences, one should calculate all the potential factors for each candidate oligonucleotide such as length, location, specificity, hairpin potential, mRNA secondary structure, and dimer formation.

Animals↗
PubMed1997

Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer.

We constructed a "cDNA bank" of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients' tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 degrees C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.

Actins↗
PubMed1997

Matrilysin gene expression in sporadic and familial colorectal adenomas.

We examined the expression of matrilysin mRNA in sporadic and hereditary colorectal adenomas to clarify the role of matrilysin in tumorigenesis. Matrilysin mRNA was not detected in normal colorectal mucosa from patients with either sporadic or familial adenomas. Matrilysin mRNA expression in sporadic adenomas correlated with the degree of dysplasia and the size of the mass, whereas most of the adenomas in patients with familial adenomatous polyposis coli expressed matrilysin mRNA irrespective of adenoma size or degree of dysplasia. Because matrilysin is more likely to be expressed in adenomas with a potential for malignancy, this enzyme may play a role in the malignant conversion of colorectal adenomas.

Actins↗
PubMed1997

[Home parenteral nutrition (HPN) for cancer patients].

We have treated thirty-five cancer patients with HPN since 1987. This paper describes its efficacy and problems. Their average age was 62 years old. The duration of HPN varied from 31 to 573 days (mean: 167 days). The catheter was removed in three cases because of catheter fever or obstruction. Metabolic complications arose in three cases, but they were easily treated. Twenty-three cases underwent pain control. Twelve cases were given cancer chemotherapy. We concluded that HPN for recurrent cancer patients is a relatively safe and effective means to improve the quality of life. We consider the following important in order to carry out HPN more smoothly. 1) Improvement of drug delivery system. 2) Establishment of home care system with other hospitals, clinics, and home care companies. 3) The easing of health-insurance restrictions on making out prescriptions to outpatients.

Adult↗
PubMed1996

Inhibition of cytochrome P450 1A1 by antisense phosphorothioate oligonucleotide in Hepa lclc7 cells.

The influence of an antisense phosphorothioate oligonucleotide has been investigated on 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A1 protein in wild type mouse hepatoma Hepa lclc7 (Hepa-1) cells. The results show that administration of a 15-mer antisense phosphorothioate oligonucleotide in ribonucleoside-free minimum essential medium effectively inhibited UV-oxidized tryptophan-inducible EROD activity and CYP1A1 protein. The inhibition of EROD activity was dose- and time-dependent. The inhibition of oxidized tryptophan-inducible EROD activity after administration of 5 microM antisense oligonucleotide for 18 hours was 74% over the control oligonucleotide-administered cells. There was no effect of the control or antisense oligonucleotide on the cell growth. This is the first demonstration that inducible CYP1A1 can be effectively inhibited by antisense phosphorothioate oligonucleotide in Hepa-1 cells. Utility of this approach should be useful in elucidating the role(s) of CYP1A1 in chemical carcinogenesis.

Animals↗
PubMed1996

Matrilysin is associated with progression of colorectal tumor.

Matrilysin and gelatinase A, B mRNA expressions were examined in colorectal tumors. Matrilysin mRNA was observed exclusively in tumors, while the others were also found in normal mucosa surrounding tumors. Further analysis revealed that colorectal adenomas with severe dysplasia, not with mild dysplasia, expressed matrilysin with lower levels than cancers. The level of matrilysin mRNA expression increased with the advancement of stages of colorectal cancers, consequently a relatively higher expression was observed in liver metastatic tumors than primary tumors. These results suggest that matrilysin mRNA expression was correlated with the progression of colorectal tumors, and this enzyme may also play a role in developing metastatic tumors in liver.

Adenoma↗
PubMed1996

Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences.

Although oligonucleotides can be easily synthesized and used in a variety of scientific fields, a major problem exists for each application: the difficulty of obtaining optimal oligonucleotide sequences. Oligonucleotide sequences have been described in each publication; however, little is disclosed on how to design such sequences and how specific each sequence is. This report introduces a new concept of computer hybridization simulation based on "thermodynamic hybridizability", which can overcome the problems of conventional homology analyses. Then, all the necessary components and factors for designing optimal probe sequences, such as hybridization strength, specificity, secondary structure, length of probes, probe-to-probe interaction, are discussed in detail. Also included are procedures for manipulating various types of data for selection of optimal oligonucleotides. This report provides a general guideline for optimal probe design and encourages basic and clinical scientists to enhance their research activities by using optimal oligonucleotides.

Base Sequence↗
PubMed1996

Technical report: Part 2. Basic requirements for designing optimal PCR primers.

Designing optimal polymerase chain reaction (PCR) primer sequences is one of the critical factors for successful PCR with sensitive, specific, and assay-to-assay reproducible results. In this review, all the requirements of PCR primer sequences are summarized, such as location, size of amplicon, length of primers, nucleotide composition, Tm, 3' terminal hybridization strength and frequency, hairpin formation energy, primer-to-primer interaction, specificity, and location of mismatches to sequences of cross-hybridization. The report also discusses how to explore these various types of information for more advanced PCR applications, which include nested PCR, multiplex PCR, competitive PCR, long PCR, point mutation detection, degenerate primers, and PCR cloning.

DNA Primers↗
PubMed1996

Software to determine optimal oligonucleotide sequences based on hybridization simulation data.

In the design of oligonucleotide sequences for targeting DNA or RNA sequences, it can be difficult to identify sequences that will hybridize only to the intended target. The term "sequence-specific" or "sequence-nonspecific" is often used to describe the interactions of an oligonucleotide with a mixture of DNA or RNA. Our new computer program, HYBsimulator (formerly OligoProbe DesignStation), creates a set of candidate oligonucleotides from a target gene. For each of the candidate oligonucleotides, a large sequence database is searched for sequences that will hybridize to the oligonucleotide. This is referred to as computer hybridization simulation (CHS). Using the nearest-neighbor model, the HYBsimulator takes into account mismatches in hybridization and calculates the melting temperature (Tm) or free energy for hybridization to all sequences in a database. The specificity of each oligonucleotide is then quantified by the number of genes that may hybridize and the predicted Tms or free energies of hybridization to those genes. The CHS data are used to select oligonucleotides based on their specificity with respect to a database.

Base Sequence↗
PubMed1996

Laboratory diagnosis of Helicobacter pylori infection by polymerase chain reaction.

Although the detection of Helicobacter pylori (Hp) infection is important, it is a rather difficult task using only conventional diagnostic techniques. In this study, we have applied a polymerase chain reaction (PCR)-based assay for the diagnosis of Hp infection. RT-PCR for Hp ribosomal RNA was utilized, using the gastric biopsy specimens obtained endoscopically from 10 patients. PCR primers have been designed from the highly consensus region of the 7 different Hp rRNA sequences in GenBank. In the detection of Hp, PCR was more sensitive than culturing and showed 100% of the sensitivity and specificity compared with histology. Our primer pair could detect as few as 50 organisms per reaction. RT-PCR for Hp rRNA is a sensitive and specific method to detect Hp infection in the gastric mucosa.

Base Sequence↗
PubMed1996

Suppression of c-jun by antisense oligonucleotides inhibits cell adhesion but not respiratory burst during phorbol ester-induced differentiation of U937 human monoblastic cells.

We studied the role of the immediate early gene c-jun in cell proliferation and phorbol 12-myristate 13-acetate (PMA)-induced differentiation in U937 human monoblastic cells, using c-jun-specific antisense (AS) phosphorothioate oligonucleotides. In selecting the most specific and potent oligonucleotide sequence, we performed extensive analyses for the binding specificity between all candidates of c-jun AS oligonucleotides and the whole sequences in GenBank database, using a computer program. Among the 20 selected oligonucleotides, two potent 15-mer AS oligonucleotides (C-JUN AS oligonucleotides) exhibited significant inhibition of cell growth in a dose-dependent manner between 2 and 10 microM. Reverse transcription-PCR and Western blot analysis demonstrated that 10 microM of C-JUN AS oligonucleotides reduced c-jun expression at both the mRNA and protein levels. More importantly, C-JUN AS oligonucleotides showed distinct effects on two markers of PMA-induced differentiation; the C-JUN AS oligonucleotides inhibited cell adhesion, whereas they did not affect another marker of differentiation, respiratory burst (measured by nitro blue tetrazolium reduction assay). These results suggest a critical role of c-jun in both cell proliferation and PMA-induced cell adhesion but not in PMA-induced respiratory burst in U937 cells.

Base Sequence↗