The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest that Thermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998) Nucleic Acids Res. 26, 4153-4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced in Escherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.

Adenosine Triphosphatases↗

PubMed2000

Evidence for early aging in the mucosal immune system.

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 microg of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21. An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level. When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4(+) T cells from spleen and Peyer's patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s. c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice. These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.

Adjuvants, Immunologic↗

PubMed2000

Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation.

RecA protein is capable of forming homo-oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A. The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.

Crystallography, X-Ray↗

PubMed2000

Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8.

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3'- and 5'-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn(2+) ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc finger and the helix-two-turns-helix motifs) suggest that the oxidized base is flipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Amino Acid Sequence↗

PubMed2000

Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA.

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

Adenosine Triphosphatases↗

PubMed2000

A novel rapid colorimetric assay of carcinoembryonic antigen levels in the abdominal cavity to detect peritoneal micrometastasis during gastric cancer surgery.

We showed that the carcinoembryonic antigen (CEA) level in a peritoneal washing is an indicator of the postoperative survival of gastric cancer patients. On the premise that the polyvinylidine difluoride membrane adsorbs a fixed quantity of protein, this study was designed to produce a rapid, colorimetric, semi-quantitative assay of peritoneal CEA levels by using anti-CEA antibodies. At the time of laparotomy, peritoneal washings were collected from 60 gastric cancer patients, and CEA levels were determined by our assay and by an enzyme immunoassay (EIA) method. The accuracy of our method corresponded with the results of the EIA method. All the cases with high levels of CEA in the peritoneal washings showed positive color changes. Our new assay had no relation to protein concentrations of the samples. The assay makes use of diluted peritoneal washings without adaptation by protein concentration. The measurement can be completed in the operating room within 30 min. As a result, this assay can detect peritoneal microdissemination easily during surgery, and it can be used as an indication of intraoperative chemotherapy against peritoneal micrometastasis.

Abdomen↗

PubMed2000

Solitary brain metastasis from papillary thyroid carcinoma in a patient with depression: report of a case.

Papillary carcinoma of the thyroid is a common thyroid malignancy with a relatively good prognosis. However, distant metastases may develop and become threatening, particularly to older patients, in a more aggressive manner. We report herein the clinical, radiological, and pathological findings of a patient with papillary thyroid carcinoma who had a solitary cerebral metastasis. The patient had been suffering from depression and had already undergone a hemithyroidectomy for primary thyroid carcinoma, and was known to have metastatic thyroid carcinoma of the lungs and bone. After the removal of the remnant thyroid gland prior to radioiodine (131I) therapy, he developed additional problems related to depression. Electroencephalography played an important role in identifying suspected brain metastasis and computed tomography demonstrated a space-occupying lesion in the left cerebral hemisphere. Consequently, an early removal of intracranial mass could be performed without any further life-threatening complications. Moreover, after removal of the brain mass the patient's depression improved immediately without the use of any antidepressants. This case report indicates the possibility that a patient's depression might be associated with brain metastasis from papillary thyroid carcinoma, and also suggests that an early diagnosis with the appropriate surgical management of a brain metastasis followed by radioiodine therapy could be valuable for achieving a prolonged disease-free period.

Bone Neoplasms↗