Biomedical subjects

M Ikeguchi

Publications and source records attributed to M Ikeguchi.

At least 109 records · Page 6Linked to original sources

PubMed1993

Patients with multiple primary gastric cancers tend to develop second primaries in organs other than the stomach.

The multiple occurrence of a single cancer tends to result in the increased vulnerability of some other organs to another malignancy. In the present study, we examined a total of 1,606 patients with gastric cancer to ascertain whether the frequency of second primaries in organs other than the stomach is higher in patients with multiple gastric cancers than in those with a single gastric cancer. Primary cancers of other organs were present in 87 (5.9%) of 1,474 patients with single gastric cancer and in 16 (12.1%) of 132 patients with multiple gastric cancers. The difference was statistically significant. The most frequent site of the occurrence of a second primary was the colo-rectum, followed by the urogenital organs, uterus, breast, and lung in descending order of frequency. We therefore assume that the occurrence of multiple cancers in the stomach is not merely a chance phenomenon but is related to both genetic predisposition and environmental factors.

Adenocarcinoma↗

PubMed1993

Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells.

Considerable evidence has accumulated indicating that overexpression of P-glycoproteins encoded by the multidrug-resistance (mdr) genes is responsible for the development of collateral resistance to a number of structurally and functionally dissimilar cytotoxic compounds in animal cells. There are three mdr genes (mdr1, mdr2, and mdr3) in the mouse genome and two (MDR1 and MDR2) in the human genome; however, only two mouse genes (mdr1 and mdr3) and one human gene (MDR1) can confer multidrug resistance upon transfection into otherwise drug-sensitive cells. Using RNase protection assay we report here that the steady-state levels of mdr1 and mdr3 messenger RNA were elevated in mouse hepatoma cells treated with dexamethasone (Dex); whereas no induction of mdr2 gene was found. Western blot analyses using anti-mdr1 and anti-mdr3 antibodies revealed that the encoded proteins appeared to be increased, but at much reduced levels. The induction was time and Dex concentration dependent. Nuclear run-on experiments demonstrated that the induction was at least in part by transcriptional control. The induction apparently required new protein synthesis since no increases in mdr1 and mdr3 transcripts was found when cultured cells were simultaneously treated with Dex and cycloheximide. Neither mdr1 nor mdr3 gene was induced in the Dex-treated nonhepatoma cell lines, LMtk- and NIH3T3. Similarly, MDR1 messenger RNA levels were elevated in the Dex-treated human hepatoma line, HepG2, but not in the nonhepatoma, HeLa. This study demonstrated that the hormonal regulation of mdr gene expression is gene and cell type specific.

3T3 Cells↗

PubMed1992

Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states.

The unfolding and refolding of a derivative of alpha-lactalbumin, in which the disulfide bond between Cys6 and Cys120 is selectively reduced and S-carboxymethylated, are investigated by equilibrium and kinetic circular dichroism measurements. The native conformation of this derivative is known to be essentially identical to that of intact alpha-lactalbumin. The equilibrium unfolding of the derivative involves a stable intermediate, which is also similar to the molten globule state of the disulfide intact protein. The results of stopped-flow circular dichroism experiments show that the same intermediate is formed rapidly as a transient intermediate in kinetic refolding. The conformational stabilities for the native and intermediate states have been estimated and compared with the stabilities for the corresponding states of intact alpha-lactalbumin. The stabilization of the native state by the disulfide has been interpreted in terms of a decrease in chain entropy in the unfolded state and elimination of the strain imposed on the disulfide bond in the native state. The molten globule state is also stabilized by the disulfide bond, although the degree of stabilization of the molten globule state is smaller than of the native state. The results suggest that, in the molten globule state, some ordered structures are present within the loop moiety formed by the 6-120 disulfide.

Animals↗

PubMed1992

Activation of multidrug resistance (P-glycoprotein) mdr3/mdr1a gene during the development of hepatocellular carcinoma in hepatitis B virus transgenic mice.

The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter. These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte. Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma. The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry. RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease. Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA. Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma. No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed. Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model. The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

[Surgical treatment of gastric cancer in the aged].

The present status of surgical treatment for gastric cancer in the aged was reviewed. The incidence among the aged in the overall operated patients with gastric cancer has increased yearly and recently has reached approximately 35%. The incidence of early gastric cancer in the aged has increased 40% in recent times. Pulmonary failure and anastomotic leak of the alimentary tract are the most common post-operative complications in the aged; these easily lead to the development of multiple organ failure with high mortality. Precise pre-operative evaluation of functional disorder in each organ and pre-, post-operative nutritional and transfusional support is indispensable to overcome these complications. Surgeons are required to perform appropriate surgical maneuvers with minimum operation time and minimum blood loss with full consideration for the adequate surgical indication in accordance with both the spread of the cancer and the general condition of the aged patient. Mental care to avoid post-operative mental disorders is also crucial.

Aged↗

PubMed1991

Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site.

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond.

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.

Chemical Phenomena↗

PubMed1989

Contribution of disulfide bonds to stability of the folding intermediate of alpha-lactalbumin.

The secondary structure formed in disulfide reduced alpha-lactalbumin is investigated by CD spectrum and is compared with that of the folding intermediate of the disulfide intact protein. The peptide backbone structure of the reduced protein depends strongly on salt concentration in contrast to that of the intermediate. It is close to a random coil in the absence of salt, but it is almost the same as that of the intermediate at a high concentration of salt. The secondary structures of both the proteins undergo broad unfolding transitions when temperature is raised or when urea is added. The secondary structure of the reduced protein is less stable against both heat and urea. These results show that the disulfide bonds are not a determinant of the secondary structure formed at an early stage of folding, and they stabilize the secondary structure of the folding intermediate.

Circular Dichroism↗

PubMed1988

A comparison of pelvic retroperitoneal pneumography and computed tomography in the assessment of extramural invasion of rectal carcinoma.

Pelvic retroperitoneal pneumography (PRP) and pelvic computed tomography (CT) were performed on 33 patients with rectal carcinoma in order to compare the usefulness of the two diagnostic procedures in the preoperative assessment of local malignant extramural invasion. Six PRP-negative patients in whom no free air was visualized in the retroperitoneal space surrounding the mass, were all assessed as having extramural invasion by CT scan and all had histologic evidence of invasion. Of 27 PRP-positive patients in whom free air was seen surrounding the mass, 18 were diagnosed as having extramural invasion on CT, 15 of whom had histologic proof of invasion. In the remaining 9 PRP-positive patients, there was no evidence of extramural invasion on the CT scans, but 5 patients showed evidence of invasion histologically. PRP, when positive, had an unacceptably high rate of being false positive and was therefore unreliable in assessing extramural invasion, whereas CT was able to detect, to some extent, extramural invasion which PRP failed to demonstrate. Based on these findings, we conclude that CT is more useful than PRP in the preoperative assessment of extramural invasion of rectal carcinoma, but is of limited diagnostic value when negative.

Adult↗

PubMed1987

High-performance liquid chromatographic-fluorescence determination of human faecal bile acids.

A high-performance liquid chromatographic method with fluorescence detection has been developed for the determination of human faecal bile acids, especially free faecal bile acids. Faecal bile acids were extracted using an Amberlite XAD-2 column and then fractionated into four groups (free, glycine-conjugated, taurine-conjugated and sulphated bile acids) on a piperidinohydroxypropyl Sephadex LH-20 column. The free bile acid fraction and free bile acids obtained after enzymatic hydrolysis and/or solvolysis of the three other fractions were derivatized with 1-bromoacetylpyrene and dicyclohexyl-18-crown-6-ether. The derivatized bile acids were separated stepwise on a Shim-pack CLC-ODS column using acetonitrilemethanol-water (100:50:30) (A), (100:50:20) (B), and (100:50:0) (C) as mobile phases with changing automatically from A to C using a solvent changer. Calibration curves of bile acid standards were linear in the range between 20 and 400 pmole, when monitored at 370 nm (excitation) and 440 nm (emission). Percent recoveries of bile acids from human faecal samples were between 80 to 95%. The method is applicable to clinical use and is sensitive, reliable, and useful for the detailed determination of human faecal bile acids, especially of free faecal bile acids.

Adult↗

PubMed1986

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme.

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.

Guanidine↗

PubMed1986

Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin.

The binding of EGTA and EDTA to alpha-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca2+ to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and alpha-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of alpha-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of alpha-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Ca2+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of alpha-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca2+ and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X 10(9) M-1 after corrections for binding of Na+ to the protein and for ionic strength, and this shows excellent agreement with the corresponding value previously estimated (2.9 +/- 1.0 X 10(9) M-1), although the latter value was obtained in the presence of EDTA. The apparent Ca2+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 X 10(8) and 8.7 X 10(6) M-1 at 0.01 and 0.1 M Na+, respectively.

Buffers↗

PubMed1986

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin.

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.

Animals↗

PubMed1986

[Types of surgical operations and cancer development].

Experimental and clinical studies were undertaken to examine whether carcinogenesis in particular organs is influenced by types of surgical operations resulting in an alteration of bile acid excretion and metabolism. A higher incidence of remnant gastric carcinoma was noted in rats receiving gastrectomy with a greater amount of duodenogastric reflux. Furthermore, carcinogenesis was enhanced in gastrectomized rats that had been fed on a high-cholesterol diet causing an increase in bile acid excretion. In a human study, however, there was no relationship between gastrectomy and the incidence of remnant stomach carcinoma. Intestinal surgery alters bile acid metabolism, which may promote colon carcinogenesis. In patients receiving ileocecal resection or right hemicolectomy, the amount of total fecal bile acids was markedly increased as compared to the normal controls. A retrospective study on the incidence of metachronous colorectal cancer in our department showed that the incidence was 12.2% in patients with ileocecal resection in contrast to 4.4% in those with left-side colon resection. These findings suggest that there is a relationship between previous ileocecal resection and the development of metachronous colon carcinoma. We could not obtain any evidence of cholecystectomy having an increased risk for the development of colorectal carcinoma.

Animals↗

PubMed1985

Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin.

Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

[Metachronous hematogenous metastasis of gastric cancer--histopathologic characteristics of gastric cancer developing of the liver, lung or bone metastasis].

Fifty-seven gastric cancer patients developing metachronous hematogenous metastasis were histopathologically analyzed. In 37 patients who developed metachronous liver metastasis as the main pattern of recurrence, papillary adenocarcinoma was the most frequent histologic type, followed by poorly differentiated adenocarcinoma of the medullary type. In 20 patients with metachronous lung or bone metastasis, poorly differentiated adenocarcinoma of the scirrhous type was the common histologic pattern. From the present data, we assume that liver metastasis of gastric cancer occurs when cancer cell clusters are transported to the liver, where they attach and proliferate, whereas single cells passed through the portal system tend to establish secondary deposits in the lung or bone.

Bone Neoplasms↗

PubMed1983

Fecal bile acids and neutral sterols in Japanese with large bowel carcinoma.

We compared the level of fecal bile acids and neutral sterols in 15 Japanese with large bowel carcinoma and 10 healthy controls. There was no significant difference in the amount of total fecal bile acids and neutral sterols between the patients and the controls. However, the amount of primary bile acids, especially chenodeoxycholic acid, tended to be higher in the patients. Conversely, lithocholic, beta-lithocholic and 3 alpha-hydroxy-12-ketocholanoic acid levels were higher in the controls. Trace amounts of 3 alpha-hydroxy-6-ketocholanoic acid were detected in some of the patients; none was found in the controls. Although a hasty conclusion should be avoided in the present study, it is of considerable interest that there was a difference in fecal bile acid composition between large bowel carcinoma patients and healthy controls.

Aged↗

PubMed1983

Lymph node removal for advanced gastric cancer with special reference to peritoneal metastasis.

The 5-year survival rate of gastric cancer patients was evaluated in relation to cancer infiltration to the gastric serosa, lymph node metastasis and lymph node removal, the pattern of cancer recurrence, and the presence of free cancer cells in the peritoneal cavity. The 5-year survival of patients with cancer infiltration to the gastric serosa was not improved even by more extensive lymph node removal (R3 removal) than secondary lymph node removal (R2 removal). In these patients, viable free cancer cells were detected in the peritoneal cavity. In patients with cancer recurrence, peritoneal metastasis was often present within 2 years of curative surgery. Based on our present results, we suggest that in advanced gastric cancer with marked serosal invasion, selective removal of more distant lymph nodes than secondary lymph nodes according to the location of the primary cancer lesion should be carried out in addition to R2 removal and that this selective removal is superior to uniform R3 removal.

Humans↗