Hydrogen-bonded complexes of 2-pyridone with centrosymmetric and non-centrosymmetric dicarboxylic acids.

2-Pyridone (2-oxopyrimidine) forms hydrogen-bonded complexes with dicarboxylic acids, the molar ratio of 2-pyridone/dicarboxylic acid being 2:1 for the complexes with oxalic acid (ethanedioic acid), 2C(5)H(5)NO.C(2)H(2)O(4), (I), and trans-beta-hydromuconic acid (trans-hex-3-enedioic acid), 2C(5)H(5)NO.C(6)H(8)O(4), (II), and 1:1 for the complexes with trans-glutaconic acid (trans-pent-2-enedioic acid), C(5)H(5)NO.C(5)H(6)O(4), (III), and L-tartaric acid (L-2,3-dihydroxybutanedioic acid), C(5)H(5)NO.C(4)H(6)O(6).H(2)O, (IV). Common features in the hydrogen-bonding patterns were found for the centrosymmetric and non-centrosymmetric acids, respectively. The 2-pyridone molecule takes the lactam form in these crystals.

A case of primary adenosquamous/squamous cell carcinoma of gallbladder directly invaded duodenum.

A rare case of primary gallbladder carcinoma is reported. A 67 year-old woman was admitted to our hospital for treatment of suspected duodenal carcinoma. A series of radiographic examinations demonstrated a giant tumor involving the duodenum, gallbladder, pancreatic head, and transverse colon. These extensions made it difficult to identify the primary origin of the carcinoma. Pancreatoduodenectomy, cholecystectomy, and resection of the transverse colon were performed. Macroscopically, ulcerative lesions were seen in both the gallbladder and the duodenum. Microscopic examination revealed adenosquamous cell carcinoma of the gallbladder, invasive of the adjacent organs, including circumferential invasion of the second portion of the duodenum. The patient tolerated the operation well and was discharged 28 days post-operatively, but died of liver metastasis 4 months after surgery. Local invasion of the surrounding tissues is characteristic of adenosquamous/squamous cell carcinoma of the gallbladder. Although surgery for cure is deemed possible, the rapid growth rate of this type of tumor may cast doubt on the value of extensive radical surgery.

Plaque formation by and plaque cloning of Chlamydia trachomatis biovar trachoma.

A new technique for the induction of plaque formation by Chlamydia trachomatis biovar trachoma applicable to the titration of infectivity and cloning of biovar trachoma was established. Three novel strains were cloned and confirmed to be free of glycogen inclusions. The lack of glycogen accumulation correlated with the absence of a 7.5-kb plasmid, which is highly conserved in other strains of C. trachomatis. Although the growth efficiency of these plasmid-free strains was slightly lower than that of plasmid-positive strains, possession of the plasmid and glycogen accumulation were not essential for the survival of C. trachomatis.

[Assay of specific anti-Chlamydia pneumoniae antibodies by ELISA method. 2. studies on clinical usefulness and serological diagnostic standards].

We measured anti-Chlamydia pneumoniae (C. pneumoniae) specific antibody titers by means of a newly-developed enzyme-linked immunosorbent assay (ELISA) method using an anti-C. pneumoniae specific antibody detection reagent. The clinical usefulness of this method was hereby evaluated. The IgG, IgA and IgM titers in 418 serum specimens obtained from patients with respiratory tract infections were measured by this new ELISA method, and the results were compared with the titers determined for the same specimens with the micro immunofluorescence (Micro-IF) method. The results showed good correlation coefficients for IgG, IgA and IgM. The two assay methods showed high agreement rates for positivity and for negativity. Specimens which did not yield the same results with the ELISA method and the Micro-IF method were subjected to analysis by the Western blot method, and the rates of agreement with the ELISA results were high. In addition, the child (0 approximately 15 yrs old; n = 122) and adult (16 approximately 90 yrs old; n = 133) cases were classified on the basis of being antigen-positive or antigen-negative at the initial examination, and their antibody-positive rates were determined. The adults showed no statistically significant differences in the antibody-positive rates for either IgG or IgA antibodies as a function of the pretreatment antigen status. However, the children showed statistically significant (p < 0.001) differences in the antibody-positive rates for both IgG and IgA antibodies as a function of the antigen status in the antigen-positive group compared with the rates in the antigen-negative group. Furthermore, the IgM-positive rates for the children were high in the antigen-positive group compared with the rates in the antigen-negative group, and the difference was statistically significant (p < 0.001). The IgM-positive rates in the adults were also significantly (p < 0.05) different between the antigen-positive group and the antigen-negative group. The Micro-IF method was applied to 34 specimens from antigen-positive patients, and 22 specimens were found to show an IgG titer of > or = 512 or an IgM titer of > or = 16. The diagnoses of these patients were acute respiratory disease in sixteen, pneumonia in four. Application of the ELISA-method to those 22 specimens showed all of them to exhibit IgG absorbance of > or = 0.6 and IgA absorbance of 0.2. The results described above indicate the clinical usefulness of our new ELISA method for the detection of antibodies specific for C. pneumoniae. The significance of this ELISA method for serological diagnosis of C. pneumoniae infections and the criteria for diagnosis of acute infections were also discussed.

[Assay of specific anti-Chlamydia pneumoniae antibodies by ELISA method. 1. Evaluation of ELISA kit using outer membrane complex].

Studies were conducted with the goal of developing a kit for assaying anti-Chlamydia pneumoniae antibodies in human serum which would enable judging positive cases with high specificity by means of an objective numerical index. Thus, an enzyme-linked immunosorbent assay (ELISA) method employing a C. pneumoniae outer membrane complex protein was established. Elementary bodies (EB) were purified from the YK-41 strain of C. pneumoniae, and subsequent treatment with Sarkosyl, DNase and RNase yielded chlamydial outer membrane complex (COMC). COMC was employed as the antigen and immobilized on 96-well microplates for ELISA method. This ELISA method was used to test 51 serum specimens from patients who had been demonstrated to be positive for C. pneumoniae antigen (throat swab: PCR positive), and the levels of IgG, IgA and IgM antibodies were assayed. For each specimen, comparison was made with the antibody titers determined by the micro immunofluorescence test (Micro-IF method). The results showed good correlation coefficients of 0.950 for IgG, 0.852 for IgA and 0.866 for IgM. In addition, the two assay methods showed the following high agreement rates: 90.2% for IgG, 84.3% for IgA and 82.4% for IgM. Specimens which did not yield the same results with the ELISA method and Micro-IF method were subjected to analysis by Western blot method, and the rates of agreement with the ELISA results were 80% for IgG, 87.5% for IgA and 88.9% for IgM. These data indicate the efficacy of this new ELISA method. Moreover, COMC was reacted with mouse antisera to three Chlamydia species, and the mouse IgG antibody was assayed. Anti-C. pneumoniae antiserum showed the strongest reactivity, whereas weaker reactivity was shown by anti-C. trachomatis antiserum (1/32nd of the reactivity of the anti-C. pneumoniae antiserum) and anti-C. psittaci antiserum (1/4th). In addition, sera from patients infected with C. trachomatis or C. psittaci (Psittacosis) were subjected to the ELISA method using COMC from C. pneumoniae. It was found that the correlation between the ELISA and Micro-IF methods was higher in relation to the anti-C. pneumoniae antibody titer than either the anti-C. trachomatis antibody titer or anti-C. psittaci antibody titer. These findings indicate this new assay kit based on the ELISA method has high specificity for C. pneumoniae.

Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.

Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.