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Biomedical subjects

M Wada

Publications and source records attributed to M Wada.

At least 289 records · Page 16Linked to original sources
PubMed1997

Construction and characterization of a rad51rad52 double mutant as a host for YAC libraries.

RAD52 or RAD51 recombination-deficient yeast strains stabilize otherwise unstable YACs containing ribosomal DNA or the human color vision locus (Kohno et al., 1994). Thus the RAD52RAD51 pathways(s) are apparently involved in the instability of YACs containing tandem repeat loci, presumably by promoting recombination-based deletion formation. Some other genomic loci are still unstable or unrecoverable in those strains, but we now find that greater stability is observed in a rad51rad52 double mutant strain that we have newly constructed. YACs containing a highly unstable region around DXS49 or centromeric regions throw off a variety of products in single mutants, but are much more stable in the rad51rad52 strain, which could therefore provide a better host for library construction and maintenance.

Chromosomes, Artificial, Yeast↗
PubMed1997

A BALB/c 3T3-transformed cell line suitable for transfection assay of metastasis-inducing genes.

A clonal cell line, 1-1ras1000, transformed by the activated c-Ha-ras oncogene, does not form metastases after i.v. injection into mice (experimental metastasis assay). Here, we show that this cell line is useful as a recipient to detect metastasis-inducing genes, using a transfection assay. Cells (1-1ras1000) were susceptible to metastasis induction by transfection with either v-src or genomic DNA from a v-src-and v-fos-transferred highly metastatic rat cell line (SR202). The susceptibility of 1-1ras1000 cells for lung metastasis induction was suitable for a genomic transfection assay to detect a metastasis-inducing gene in the transfected cells which had incorporated genomic DNA from donor metastatic tumor cells. When DNAs extracted from 7 human tumors were tested for metastasis induction, 2 DNAs from nonmalignant tumors (non-tumorigenic tumors in athymic nude mice) (2/2) were negative and 4 DNAs from malignant tumors (4/5) were positive in 1-1ras1000 cells for primary transfection. in one of the resulting metastases, the ability to metastasize was also transferred in the second and third cycles of genomic DNA transfection at high frequencies. All of the resulting metastases carried the human repetitive Alu sequence. Neither re-arrangements of the endogenous c-Haras nor changes of protein amounts were detected. Recipient 1-1ras1000 cells had a negligible rate of spontaneously metastatic conversion during in vitro cultivation and transfection processes. The resulting metastasized cells were easily isolated from the lung after culturing in selection medium containing G418 (geneticin). Isolated cells stably retained the ability to form metastatic lung nodules when re-injected into mice. Thus, 1-1ras1000 cells appear to be a useful system for the isolation of metastasis-inducing genes from human metastatic tumors.

3T3 Cells↗
PubMed1997

Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins.

The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). No GEP specific for each Rab family member or Rab subfamily has been isolated. Here we purified a GEP from rat brain with lipid-modified Rab3A as a substrate. The purified protein was specifically active on Rab3A, Rab3C, and Rab3D of the Rab3 subfamily. Of these subfamily members, Rab3A and Rab3C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GEP (Rab3 GEP) was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GEP showed a minimum molecular mass of about 200 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and determined its primary structure. The isolated cDNA encoded a protein with a Mr of 177,982 and 1,602 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GEP activity toward Rab3A, Rab3C, and Rab3D. Northern blot and Western blot analyses indicated that Rab3 GEP was expressed in all the rat tissues examined with the highest expression in brain.

Amino Acid Sequence↗
PubMed1997

Effects of day length and temperature on gonadal development, body mass, and fat depots in white-crowned sparrows, Zonotrichia leucophrys pugetensis.

We tested the effects of ambient temperature (5 degrees, 20 degrees, and 30 degrees) on photoperiodically induced reproductive functions in male and female white-crowned sparrows, Zonotrichia leucophrys pugetensis. Transfer from short days (9L 15D) to long days (16L 8D) resulted in rapid testicular growth and partial ovarian development in all three temperature treatments. There were no differences in sizes of testes and cloacal protuberance following 30 or 70 days of exposure to long days at the different temperatures. However, brood patch and follicular development were enhanced in females at 30 degrees compared with the 5 degrees and 20 degrees groups. Many of these females exposed to 30 degrees had large yolky follicles by Day 70. This enhancement was evident only when females were housed in the same room with males, however. Despite the effects of high temperature on ovarian development, there were no differences among groups in plasma levels of follicle-stimulating hormone or luteinizing hormone, suggesting that differential ovarian development may have been mediated by gonadal sensitivity to gonadotropins rather than by differential secretion of these hormones. We examined circulating levels of corticosterone (B) and both tri-iodothyronine (T3) and thyroxine (T4) as possible regulators of this differential ovarian sensitivity to gonadotropins. Plasma B levels showed transitory increases in males at 5 degrees and 20 degrees, but were suppressed in males at 30 degrees. Titers of B were not influenced by temperature treatments in females. Circulating T4 increased following photostimulation in both sexes, but this increase was reduced at 5 degrees. T3 concentrations in plasma were highly variable and not influenced by either photo-period or temperature in males, but were significantly lower in females exposed to 30 degrees by Day 70. Thus, B and T4 levels do not appear to help explain differential ovarian development, but circulating T3 levels cannot yet be excluded as a regulator of ovarian sensitivity to gonadotropins. Long days resulted in no change, or a gradual decrease, in body mass and fat deposit in males and females, and temperature regimes had no further effects on fattening or body mass. Thus, reproductive development under long days appears to be resistant to naturally relevant temperature extremes in male Z.l. pugetensis, whereas follicular development (i.e., yolk deposition in follicles leading to ovulation and onset of nesting) can be enhanced by high temperature. Reasons for the dimorphism in this response are unknown, but may be explained by the role of females in determining onset of final ovarian maturation and nesting in relation to favorable environmental conditions. In a second experiment, in which the sexes were isolated from one another, we determined the effects of the same treatments on.Z.I. pugetensis. Again there was no effect of temperature on photoperiodically induced testicular growth, and the enhancement of follicular development in females at 30 degrees was greatly reduced in the absence of males. We also continued this experiment up to 116 days of treatment to investigate effects on onset of photorefractoriness (spontaneous gonadal regression) and onset of prebasic moult. In both sexes it was clear that low temperature (5 degrees) retarded gonadal regression and high temperature (30 degrees) advanced it. Similarly, the prebasic moult score was greater at 30 degrees and less at 5 degrees in both sexes. There were no effects of temperature on plasma levels of LH at Day 116 of treatment, but plasma levels of T4 were higher in the 5 degrees group of both males and females sampled at Day 116. Clearly, the effects of temperature can have different effects on gonadal recrudescence, onset of breeding (yolk deposition), and termination of breeding. Whether these influences of temperature on reproductive function at different stages in the breeding cycle have different mechanisms remains to be determ

Adipose Tissue↗
PubMed1997

Ischemia-reperfusion injury on the pancreas in rats: identification of acinar cell apoptosis.

An ischemia-reperfusion injury on the pancreas is involved in the pathophysiology of acute pancreatitis or tissue injuries after pancreas transplantation. On the other hand, recent studies have demonstrated that ischemia-reperfusion induces apoptosis in several organs such as kidney, heart, and brain. In the present study, we sought to characterize a pattern of injury during ischemia-reperfusion on the pancreas and determined whether ischemia-reperfusion on the pancreas causes the apoptotic process. Ischemia-reperfusion was induced by blocking the inferior splenic artery and removing the clamp in pentobarbital-anesthetized rats. Rats were sacrificed at 0-72 hr following a 60-min ischemia. Evans blue extravasation showed 3.5-fold increase at 2 hr after reperfusion, indicating a rapid increase of vascular permeability. Tissue myeloperoxidase activity, an index of neutrophil accumulation, significantly increased in a time-dependent manner until 48 hr after reperfusion. Histological analysis revealed the existences of interstitial cell infiltration and edema. DNA breaks of acinar cells were detected by gel electrophoresis and in situ nick end-labeling, and the numbers strikingly increased at 48 hr after reperfusion. Furthermore, Bax protein, an effector of apoptotic cell death, was expressed in acinar cells. The results indicate that an ischemia-reperfusion injury on the pancreas in rats resembles many features of acute pancreatitis. Apoptosis in acinar cells may be one of the specific features of the ischemia-reperfusion injury on the pancreas.

Amylases↗
PubMed1997

Detection of matrix metalloproteinase activity in human pancreatic cancer.

Destruction of the basement membrane (BM) is mandatory for tumor spread, and matrix metalloproteinases (MMPs) are known to be implicated in colon cancer invasion and metastasis by digesting type IV collagen, a main component of the BM. The current study analyzed the expression of MMP-2 and MMP-9 in pancreatic cancer tissues. Frozen specimens of pancreatic cancer (n = 10), a liver metastatic nodule from pancreatic cancer (n = 1), and normal pancreas (n = 3) were homogenized and analyzed by zymography. The activated form of MMP-9 (82 kDa) was detected in all of the normal and malignant tissues, while the activated form of MMP-2 (62 kDa) was detected in all of the pancreatic cancers and its metastatic tissue, but not in the normal pancreatic tissues. These results indicate that expression of the activated form of MMP-2 may be specific to pancreatic cancer, while that of MMP-9 may be unrelated to it.

Biomarkers, Tumor↗
PubMed1997

Chemosensory-event-related potentials to olfactory stimulations.

Odorant pulses were introduced into the noses of 18 healthy volunteers by using a pressurized nasal spray. Just prior to the onset of inspiration, the spray tip was inserted 1 cm into the nostril as atraumatically as possible. The odorant pulse trigger was the subject's respiration. Responses to aerosolized skatole consisted mainly of a positive wave with a peak latency of 147.1 +/- 5.3 ms. These positive waves were distinguishable as an evoked response by employing curve superimpositions before and after averaging positive responses. In our cases, saturation of responses was found after 4-5 averagings with the response becoming most clear after 7-8 averagings. The response peak latency to more concentrated skatole was consistently shorter by 9-19 ms than that to 10% diluted skatole in all subjects tested.

Administration, Intranasal↗
PubMed1997

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Exacerbated autoimmune hepatitis successfully treated with leukocytapheresis and bilirubin adsorption therapy.

A 58-year-old man with subacute fulminant onset of autoimmune hepatitis (AIH) was treated by leukocytapheresis (LCAP) and bilirubin adsorption therapy (BAT), rather than by administration of high-dose corticosteroids as he had mild glucose intolerance, and a definitive diagnosis of AIH was not obtained on admission; further, there was a risk of viral infection. After initiation of the therapies, serum transaminases and bilirubin, immunoglobulins, anti-nuclear antibodies, and rheumatoid factor decreased rapidly, as did the initially high levels of activated cells and several pro-inflammatory cytokines. Liver inflammation observed on liver biopsy settled during the course of the therapies, with no adverse side effects. A pause in the therapies was associated with deterioration; however, restoration of apheresis was followed by normalization. Remission was sustained throughout the period monitored, except for a recurrence 14 months after discharge, which was successfully resolved by two additional LCAP sessions. These results suggest that LCAP influences the causal mechanism(s) of exacerbation of AIH.

Adsorption↗
PubMed1997

Molecular analysis of the adenomatous polyposis coli gene in sarcomas, hematological malignancies and noncolonic, neoplastic tissues.

Somatic mutations of the adenomatous polyposis coli (APC) gene have been frequently found in sporadic colorectal tumors, and the frequency of such mutations remain constant as tumors progress from benign adenomas to malignant cancers. Thus the mutations of the APC gene may have a major role in the early development of sporadic colorectal tumors. Whether inactivation of the APC gene accounts for other types of primary tumors is still being investigated. We investigated for APC mutations within the mutation cluster region (a 684-bp region containing most of the mutations found in colorectal tumors) in 317 samples from a wide variety of human malignant and premalignant tissues, including 40 lung cancers, 47 renal cell carcinomas, 41 osteosarcomas and 21 other types of sarcomas, 45 acute lymphoid leukemias/lymphomas, 33 acute myeloid leukemias, 27 myelodysplastic syndrome samples, and 20 chronic colitis (ulcerative colitis and Crohn's disease) associated cancers and dysplasias, and 43 human malignant cell lines. We used single-strand conformation polymorphism assay following polymerase chain reaction. Samples with abnormal assay results were reamplified and analyzed by the direct DNA sequencing method. We detected a total of two cases with a base substitution. A silent mutation was detected in a case of myelodysplastic syndrome, and a novel nonsense mutation was discovered in a colorectal cancer cell line, SW837. In summary, we did not detect any functional mutations of the APC gene in a wide variety of tumors except for a colon cancer cell line, suggesting that alterations of the APC gene do not have a major role in the development of lung and renal cancers, various types of sarcomas, or hematological malignancies.

Colonic Neoplasms↗
PubMed1997

[Are negative cognitions symptoms of depression or also an expression of personality traits?].

This study examined the changes from hospital admission to discharge of different categories of cognitive distortions - automatic thoughts, self-concepts and dysfunctional attitudes - in 67 patients with an ICD-10 diagnosis of a depressive episode. Furthermore, the relationships were analyzed between cognitive distortions, degree of depression, and personality dimensions (isolation tendency, extraversion, rigidity, schizoidia, frustration tolerance and neuroticism). There was a significant correlation between automatic thoughts and negative self-concepts on the one hand, and severity of depression on the other upon admission. There was a significant reduction of automatic thoughts and the negativity of self-concepts from admission to discharge. No significant association was observed, however, between the severity of depression and dysfunctional attitudes. In addition, dysfunctional attitudes showed a significant decrease until discharge only in patients with a full remission of depressive symptoms. These results underline the fact that automatic thoughts and self-concepts are more state-dependent than dysfunctional attitudes. Moreover - even taking into account the degree of depression - there was a significant relationship between the personality dimension neuroticism and all cognitive scales upon admission. This indicates that a high degree of neuroticism may facilitate the emergence of negative cognitions during a depressive episode.

Adult↗
PubMed1997

Enzymatic production of ethyl (R)-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast.

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.

Aldehyde Reductase↗
PubMed1997

Vertebral lesions associated with palmoplantar pustulosis.

Spinal lesions were reviewed in 9 patients (3 men and 6 women) who presented with palmoplantar pustulosis. Cervical lesions were found in 3 patients and lumbosacral involvement in 7. The duration of disease averaged 4.8 years. Seven patients had associated sterno-costo-clavicular hyperostotic lesions, and all showed abnormalities in blood tests which suggested chronic inflammation. Vertebral lesions were assessed as a spondylodiscitis type in 4, single level syndesmophyte-forming type in 1, multiple hyperostosis type in 4, and an ivory vertebra type in 1. Five patients were treated conservatively, and 4 with progressive spondylodiscitis type lesions underwent curettage and anterior interbody fusions. At follow-up, all patients improved in both spinal and cutaneous presentations, and 4 who underwent surgery and 1 with tonsillectomy attained complete cure. The vertebral manifestations of palmoplantar pustulosis may vary, but rapidly progressive lesions need surgery for favourable early improvement from the vertebro-cutaneous symptom complex.

Female↗
PubMed1997

Stress fracture of the femoral component in total knee replacement: a report of 3 cases.

We report 3 cases of stress fracture of the femoral component (small size Whiteside Ortholoc II) in total knee replacements which occurred at 32, 52, and 73 months after operation. The site was at the junction between the medial posterior bevelled surface and the posterior flange. The most likely cause of failure is due to the thinness of the metal at this point.

Aged↗
PubMed1997

The tarsal tunnel syndrome: evaluation of surgical results using multivariate analysis.

Thirty-four patients with the tarsal tunnel syndrome were treated by decompression of the posterior tibial nerve. The condition was bilateral in 3 cases. There were 9 men and 25 women with an average age at operation of 41 years. The average follow up was for 3.8 years. Multivariate analysis showed that the outcome is influenced, in order of importance, by fibrosis around the nerve, the preoperative severity of the condition, a history of sprained ankle, worker's compensation, a long history, and heavy work. The results were favourable when there was a short history, the presence of a ganglion, no sprains, and light work. Measurement of the terminal latency of the medial plantar nerve was valuable in assessing recovery. The precise cause of the syndrome and its effect on treatment should be considered before operation.

Adolescent↗
PubMed1997

Plasticity of the spinal cord contributes to neurological improvement after treatment by cervical decompression. A magnetic resonance imaging study.

To investigate the relationship between morphological plasticity of the spinal cord and neurological outcome after surgery for compressive lesions, we correlated the transverse area of the cervical spinal cord measured by transaxial magnetic resonance imaging (MRI) obtained during the early postoperative period (1-6 months) with neurological function assessed at a median postoperative follow-up period of 2.5 years. Measurements on MRI in 56 patients (35 men and 21 women) included evaluation of the cross-sectional area of the cervical cord and the subarachnoidal space at the level of decompression. The transverse area of the cervical cord increased by 30 to 62% postoperatively and that of the subarachnoidal space by 57 to 95%. Neurological improvement was noted in all patients and averaged 63% in our assessment scale. Expansion of the cervical cord during the early postoperative period correlated significantly with the late postoperative neurological status (P = 0.009). Our results suggest that an increase in the cross-sectional area of the cervical spinal cord, representing spinal cord morphological plasticity, is a significant factor in determining the late neurological improvement following decompressive surgery.

Adult↗
PubMed1997

Effects of structural modulation on biological activity of bombesin analogues with (E)-alkene bond.

The specific bombesin receptor antagonist, (E)-alkene bombesin isostere (EABI-1), [D-Phe6,Leu13psi[(E)CH=CH]Leu14]bombesin(6-14) is a potent antagonist in terms of inhibition of bombesin-stimulated amylase release from rat pancreatic acini. This study examined the effects of EABI-1 (L-L diastereomer) and three novel bombesin analogues on amylase release in rat pancreatic acini. EABI-2 is a L-D diastereomer of EABI-1. EABI-3 is an analogue, of which leucine at position 13 of EABI-1 was replaced with valine. EABI-4 is a L-D diastereomer of EABI-3 (L-L). The order of agonist potency was EABI-2>EABI-3>EABI-4. EABI-1 showed no agonist activity at concentrations up to 100nM. On the other hand, all of four analogues had antagonist activity. The order of antagonist potency was EABI-1>EABI-3>EABI-4>EABI-2. EABI-1 was a complete antagonist, EABI-2 and EABI-3 were partial agonists, and EABI-4 had a weak agonist effect. The present study provides a useful information on the future development of peptide analogues for anticancer agents and biological tools for investigating actions of bombesin family peptides.

Amylases↗