Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan.

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.

Isolation of chlamydia psittaci from domestic cats with oculonasal discharge in Japan.

Eight strains of Chlamydia psittaci were isolated in Japan from the nasal and conjunctival swabs of six household cats using the L929 cell line of mouse fibroblast origin. The isolates were identified as C. psittaci on the basis of the formation of characteristic inclusion bodies in the cell culture detected by Giemsa stain and immunofluorescence. Comparison of nucleotide sequences of the ompA gene amplified from the three isolates with the published sequence of feline FEPN strain of C. psittaci showed almost 100% homology.

Synthesis and antifungal activities of novel 1,3-beta-D-glucan synthase inhibitors. Part 2.

Highly potent 1,3-beta-D-glucan synthase inhibitors, 7b, 10a, 10b and 12, have been identified by the chemical modification of the ornithine residue of a fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. These compounds showed stronger antifungal activity against systemic candidiasis as well as pulmonary aspergillosis in mice, and less hepatotoxicity as compared with 1.

Complete nucleotide sequence of a group A avian rotavirus genome and a comparison with its counterparts of mammalian rotaviruses.

The nucleotide sequences encoding four structural proteins (VP1-4) and six nonstructural proteins (NSP1-6) of avian rotavirus PO-13 were determined. Based on the results of earlier sequencing studies [Ito et al., 1995, Sequence analysis of cDNA for the VP6 protein of group A avian rota viruses. Arch. Vriol. 140, 605-612; Rohwedder et al., 1997, Chicken rotavirus Ch-1 shows a second type of avian VP6 gene, Virus Genes 15, 65-71; Rohwedder et al., 1997, Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein, Virus Genes 14, 147-151], determination of PO-13 genome sequence has been completed. The PO-13 genome is 18845 nucleotides in length. It is 290 nucleotides longer than the genome of SA11. The amino acid sequence homology between PO-13 and mammalian rotaviruses ranged from 76-77% (VP1) to 16-18% (NSP1). The features of gene and amino acid sequence were compared with those of the corresponding protein of mammalian rotaviruses. Based on results of the phylogenetic analyses of NSP1, we speculate that an ancestral rotavirus could have separated into groups A, B and C rotaviruses at an early evolutionary stage and that group A rotavirus separated into mammalian and avian rotaviruses with host evolution.

Synthesis and antifungal activities of novel 1,3-beta-D-glucan synthase inhibitors. Part 1.

Highly potent 1,3-beta-D-glucan synthase inhibitors 10, 11 and 13 have been identified by the chemical modification of the fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. D-Ornithine derivative (10) showed improved antifungal activity in the systemic candidiasis model in mice and reduced hepatotoxicity in vitro, as compared with 1.

The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine.

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.

Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans.

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

Synthesis and structure-activity relationships of novel fungal chitin synthase inhibitors.

A novel Candida albicans chitin synthase 1 (CaChs1) inhibitor, RO-41-0986 (1) was discovered by random screening. Systematic modification led to the identification of a highly potent CaChs1 inhibitor, RO-09-3024 (2), having strong antifungal activity against Candida spp. in vitro.

2-Methoxy-4-nitroaniline and its isomers induce cytochrome P4501A (CYP1A) enzymes with different selectivities in the rat liver.

We reported previously that 2-methoxy-4-nitroaniline (2-MeO-4-NA) is a selective inducer of cytochrome P4501A2 (CYP1A2) in the rat liver, and its molecular size is the smallest among known CYP1A2-selective inducers. In the present study, a structure-activity relationship on the CYP1A2-selective induction has been investigated using isomeric nitroanisidines and their related chemicals. Western blot analyses revealed that the chemicals removed a substituent (amino, methoxyl or nitro group) from a 2-MeO-4-NA molecule had no capacity for inducing CYP1A enzymes in rat livers. On the other hand, isomeric nitroanisidines such as 2-MeO-4-NA, 2-MeO-5-NA and 4-MeO-2-NA induced both CYP1A2 and CYP1A1 enzymes with different selectivities. As judged from the induced levels of CYP1A proteins, 2-MeO-4-NA (CYP1A2/CYP1A1 ratio; 9.5) and 4-MeO-2-NA (0.3) were the most selective inducers of CYP1A2 and CYP1A1, respectively, among the isomeric nitroanisidines (0.44 mmol/kg) used. The induced level of CYP1A2 protein was in the order 2-MeO-4-NA > 2-MeO-5-NA > 4-MeO-2-NA, although no significant difference was observed on their CYP1A2 mRNA level. On the contrary, increases in the levels of CYP1A1 mRNA and protein were in the order 4-MeO-2-NA > 2-MeO-5-NA > 2-MeO-4-NA. The present findings indicate that all three substituents (amino, methoxyl and nitro groups) are necessary components of nitroanisidines for induction of CYP1A enzymes, and also show that regio-isomeric positions of these substituents determine the selectivity in the induction of CYP1A enzymes.

Human papillomavirus type 18 DNA sequences in adenocarcinoma and adenosquamous carcinoma of the uterine cervix.

Human papillomavirus (HPV) DNA sequences were detected by Southern blot hybridization and polymerase chain reaction (PCR) in 10 out of 19 patients (52.7%) with adenocarcinoma [15] and adenosquamous [4] carcinoma of the uterine cervix. HPV 18 DNA was detected in 8 of these 19 patients (42.1%), HPV 16 DNA in 1 patient (5.3%) and HPV type X (unknown) in another (5.3%). Of the 10 HPV positive samples HPV 18 was found in 6 out of 6 pure adenocarcinomas (100%), and in 2 of 4 (50%) adenosquamous carcinomas. HPV 16 and HPV X were each detected in 1 out of 4 (25%) adenosquamous carcinomas. The physical state of the viral DNA was investigated in 5 of the 10 HPV-positive cases. All the specimens from these 5 cases showed HPV to be integrated into the host genome, except for one adenosquamous specimen, which showed both episomal and integrated forms of HPV 16. Six of 8 HPV 18 DNA positive specimens were from cases of pure adenocarcinoma and it was found by PCR that five of these 6 specimens retained fragments of E6/E7, LCR/E7 and early sequence of E1 fragment (sequence: 1188-1373) but deleted most part of E1.

Studies on ras oncogene activation in endometrial carcinoma.

The frequency of K-ras point mutation(PM) at codon 12 was studied in 45 patients with endometrial carcinoma. In vitro amplification of target sequences of DNA extracted from endometrial cancer tissues by polymerase chain reaction and dot blotting with oligonucleotide hybridization were performed. Ten of 45 endometrial carcinomas disclosed K-ras PM at codon 12 (22.2%). Transition from GGT to GAT was most frequent in PM(41.7%). Simultaneously, double PM (GAT/GCT) were also detected in 2 cases. No relationship appeared to be present between PM and clinical prognosis such as clinical stage, histological type, histological grade of differentiation, depth of myometrial invasion, and ascitic cytology. The positive rates of lymph node metastasis tended to be higher in the group with positive PM than in the group without PM. K-ras and C-myc gene amplifications were found in 2 (5.1%) and 3 (7.7%) of 39 cases, respectively. No PM of H-ras at codons 12 and 61 was detected. Our results showed that the PM of K-ras gene at codon 12 was a fairly common event in genetic abnormality and suggested it would have some role in the progression of carcinogenesis in endometrial carcinoma.

Cisplatin-based chemotherapy for advanced or recurrent endometrial cancer - a review of 36 cases.

Thirty-six patients with advanced or recurrent endometrial cancer having had no previous chemotherapy were treated with cisplatin-based chemotherapy. The overall response rate was 38.9%. The mean time to response and the mean duration of response were 9.6 weeks and 16.9 months, respectively. There was no significant difference in response and survival probability between persistent disease and recurrent disease. Size of tumor had a marked influence on response, while age, history of previous irradiation and histologic grade of adenocarcinoma did not affect the response rate. Survival probability by response clearly demonstrated that only complete response exhibited a large impact on survival.

[Multimodal treatment for advanced, ovarian cancer patients with poor performance status--its effectiveness and limitations].

We assessed the feasibility of the sequential multimodal treatment including neoadjuvant chemotherapy for far advanced ovarian cancer patients not amenable to a standard modality because of poor medical status. Seven consecutive advanced ovarian cancer patients presented with massive ascites (5 with pleural effusion). Based on the priming theory, immunotherapy with OK432 (s.c. priming with 0.2 KE of OK432 followed by a local injection with 10KE of OK432) was successfully applied to a carcinomatous effusion. Thereafter, patients were treated with 4-6 courses (13-20 wks) of "low-dose consecutive CP (CPM 500 mg/m2, day 1; CDDP 10 mg/m2, day 1-7), which delivered 1CR, 5PR and 1NC (tumor regression rate: 30-100%). Subsequently, the patients underwent radical surgery including small/large bowel resection, splenectomy and diaphragma resection in addition to hysterectomy, bilateral adenectomy and omentectomy, with tumor resectability being 100% in 4 cases and 90% < (residual 2 cm) in 3. Postoperatively, patients received intraperitoneal (IP) chemo-immunotherapy and were followed up with IP washing cytology through an implanted reservoir. Mean survival time was 17.1 months (10-32) with the follow up interval being 10-32 months. Four patients with complete tumor resection are alive with no evidence of disease for 14-32 months. Among 3 incomplete patients, 2 with persistent/recurrent disease received further IP chemotherapy and the remaining one died of the disease at 15 months from the start of therapy. Thus, the present multimodality indicates the possibility of a "cure" for far advanced ovarian cancer patients with poor performance status.

Immunohistochemical and ultrastructural investigation of new membrane-associated placental tissue proteins (MP2 A, B, C, D, and E) in gynecologic neoplasms.

New membrane-associated placental tissue proteins (MP2 A, B, C, D, and E) were investigated immunohistochemically by avidin-biotin immunoperoxidase technique and immunoelectron microscopy in various gynecologic neoplasms and normal gynecologic tissues. MP2 A and MP2 B were not specific for malignant tumors. MP2 C was present in 67-100% of ovarian carcinomas, 100% of benign dermoid cysts, and 77% of endometrial carcinomas. Except for endocervical adenocarcinomas, MP2 D was hardly detectable in gynecologic malignancies. Although MP2 E was hardly detectable in benign gynecologic tumors, this protein was present in ovarian carcinomas, uterine squamous carcinomas, endocervical adenocarcinomas, and endometrial adenocarcinomas. These results suggest a possible clinical application of these MP2 proteins as a new tumor marker for gynecologic malignancies.

The immunohistochemical localization of new membrane-associated placental tissue proteins (MP2 A, B, C, D, and E) in human and cynomolgus monkey placentae.

New membrane-associated placental tissue proteins (MP2 A, B, C, D, and E) were investigated by avidin-biotin immunoperoxidase technique in the human and cynomolgus monkey placentae, decidua and umbilical cords. In human early placentae, MP2 A, B, C, and E were localized mainly in the membrane of villous syncytiotrophoblasts and cytotrophoblasts. Histiocytes in the villous stroma were positive for MP2 A, B, D, and E. In human term placentae, obvious positive staining for MP2 A, B, C, and E was observed in the membrane of villous syncytiotrophoblasts, in the amniotic epithelium, and in the umbilical cord sheath. Histiocytes in the villous stroma were positive for MP2 A, B, C, E, and especially for MP2 D. Importantly, MP2 A, C, and E were positive in polymorphonuclear neutrophils, since most of these common antigens are also carcinoma-associated, suggesting clinical usage of MP2 proteins as a new tumor marker. In the cynomolgus monkey placentae, similar immuno-staining results were obtained. The monkey can thus serve as a experimental model for the investigation of the placental proteins.

Cytomorphologic, cytometric and histomorphologic observations after laser therapy for cervical lesions.

To analyze the healing process after laser therapy for cervical lesions, the clinical, cytologic, histologic and colposcopic features in 109 cases were studied chronologically. The healing process of the cervical epithelium usually began from both the squamous and columnar epithelial borders, starting around the 10th day after laser therapy; the process covered the whole tissue defect with multilayered epithelium within seven weeks. Inflammatory changes also usually abated within that time. Cytomorphologically, laser therapy resulted in the occurrence of (mostly degenerated) "fiber-type" and orangeophilic cells in smears taken during the first two weeks after treatment. Tissue repair cells were seen in smears collected from the first posttherapy day through the fourth week after laser therapy. Using computer-assisted image cytometry, the reparative cells in samples taken shortly after treatment (roughly, the first to fifth days) exhibited more hyperchromatic (3-4N) nuclei than did those in later samples; however, the mean DNA content of the early reparative cells was generally concentrated around that of the 2N reference cells. These findings suggest that follow-up, including cytologic and colposcopic examination, for the early detection of residual or recurrent lesions should start in the eighth week and continue periodically for at least one year.

Augmenting effect of sizofiran on the immunofunction of regional lymph nodes in cervical cancer.

The immunomodulating effect of sizofiran on regional lymph nodes (RLN) was studied in cervical cancer and benign gynecologic tumors comparatively between treated patients (sizofiran intramuscularly (1) 1 day (20 mg) before and (2) 8 days (20 mg) and 1 day (20 mg) before surgery; n = 34) and untreated patients (n = 21). The RLN (internal iliac lymph nodes) were dissected surgically, and freshly obtained mononuclear (MN) cells were studied to observe interleukin-2 (IL-2) production and lymphokine-activated killer cell and natural-killer cell activities. The infiltration of surface phenotype of MN cells into RLN was determined semiquantitatively by immunohistochemistry. Sizofiran augmented IL-2 production of RLN, which was accompanied by a marked increase in the number of cells stained with anti-Leu-3a and IL-2 receptor. This effect was found more often in Stage Ib disease than in Stage 0, and it was not observed in benign tumors. These results suggest that stimulation with some antigens (e.g., cancer antigen in the current study) is necessary to induce immunoaugmentation by sizofiran which has no antigenicity. The augmenting effect was seen even in lymph nodes involved by advanced cervical cancer. Therefore, sizofiran was found to be a potent biologic response modifier in patients with cervical cancer.

Changes in bone mineral density and fracture prevalence in Japanese women after oophorectomy.

The bone changes after gynecological surgery during the early phase of recovery were examined. The subjects were randomly selected from women who had undergone bilateral oopho-hysterectomies (OOX, n = 98, 46.0 +/- 5.0 year-old) or hysterectomies (HX, n = 75, 43.6 +/- 4.6 year-old) within 4 years prior to entering the study. The ovarian functions in the HX group were presumed to be intact following the hysterectomies, judging from the cytological evaluation of the vaginal pap smears. The bone morphological changes in both groups were examined to measure the cortical thickness of the metacarpal bone (MCI) on hands X-ray film using microdensitometry, and the posterior/anterior height ratios (P/A ratio) of the entire vertebral bodies, on vertebral X-ray films using a digitizer. The changes in bone mineral densities in both groups were measured by dual energy X-ray absorptiometry at lumbar vertebrae (L2-4 BMD) and by microdensitometry at the metacarpal cortical bone; the bone densities of metacarpal bone were referenced by the density of aluminum step wedge on the same X-ray films. The prevalence of vertebral body fractures (P/A ratio greater than 1.4) in the OOX group (5.1%) was 3.9 times higher than that in the HX group (1.3%). There was a significant decrease in MCI in the OOX group compared with the HX group (p less than 0.01). L2-4 BMD in the OOX and HX group were 1.07 +/- 0.15 and 1.16 +/- 0.13 g/cm2, respectively (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

[Time course changes in bone mineral density and calcium metabolism after oophorectomy].

Time course changes in bone mineral density and calcium metabolism after oophorectomy were investigated in comparison with those in hysterectomized and pre-operated women, retrospectively. A total of 191 women who had a regular menstrual period for 1 year prior to entering the study or to the operation, were divided into 3 groups; group 1 consisted of 33 women who were admitted to receive a gynecological operation (pre-OP group), group 2 consisted of 57 women who received hysterectomy within 5 years prior to entering the study (HX group) and group 3 consisted of 101 women who received hysto-oophorectomy (bilateral) at the same timing as the HX group (OOX group). All the subjects had their bone mineral density measured at the lumbar vertebrae (L2-4BMD) by means of dual energy X-ray absorptiometry (DXA) and at the metacarpal bone by the microdensitometrical (MD) method. Serum and urinary parameters related to bone and calcium metabolism were also evaluated. After dividing the subjects in accordance with the length of time after the operation, the time course changes in each parameter were calculated. In the 2 years after OOX, the L2-4BMD decreased significantly and rapidly, and the change in L2-4BMD in the subsequent period was quiescent. In the HX group, there was no significant change in L2-4BMD. When the period after the OOX was divided into 2 subcategories in accordance with the speed of decline in lumbar BMD as the rapid and the slow phases, the time course changes in blood and urine parameters showed different trends.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-alpha, interferon-gamma and sizofiran in the adjuvant therapy in ovarian cancer--a preliminary trial.

The study included 24 cases of negative second-look laparotomy (SLL) after operation on ovarian cancer. 12 cases were treated with sizofiran and recombinant interferon-gamma before and after SLL and then with human lymphoblastoid interferon-alpha. The remaining 12 cases (controls) were followed up without any drug therapy after SLL. There were no recurrences in the treated group, but in 3 cases of the control group. Also significant difference in survival was noted in the treated group.