Biomedical subjects
Publications and source records attributed to Y Nakao.
Bioassay-guided isolation of serine protease inhibitors from a marine sponge, Coscinoderma mathewsi, has yielded 1-methylherbipoline salts of halisulfate-1 and of suvanine. Structures of these compounds were identified by spectroscopic analyses and literature data. Antithrombin and antitrypsin activity was determined.
Rat sciatic nerve graft segments were harvested and pretreated by either placement in the University of Wisconsin Cold Storage Solution at 5 degrees C and storage from 1 to 26 weeks, or repeatedly freezing (-40 degrees C) and thawing (20 degrees C). Following pretreatment, grafts were transplanted as either syngeneic or allogeneic nerve grafts. Storage and freeze-thawing did not affect the Schwann cell basal lamina or laminin distribution of the peripheral nerve. Graft cell viability decreased with increasing time of storage, with some viable cells detectable even after 3 weeks of storage. Freeze-thawed grafts were not viable. Increasing time of storage led to decreasing immune response and graft rejection, but improved regeneration. Freeze-thawed and 26-week stored allografts were nonimmunogenic and rejection was not seen, but regeneration was delayed compared to autografts. Graft storage may become a useful adjunct to clinical nerve allografting to permit elective scheduling of surgery, provide greater time for preoperative tissue testing, and possibly blunt the immune response.
The effects of the anti-voltage-gated Ca2+ channel (VGCC) antibody obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS) on autonomic neurotransmission were studied in in-vitro experiments. The releases of acetylcholine (ACh) and norepinephrine from the autonomic nerves were evaluated by changes in the contractile responses of guinea pig taenia caeci and left atria to electric field stimulation, respectively. Incubations for 6 hours with LEMS serum and IgG, both of which contain anti-VGCC antibody, markedly suppressed the parasympathetic response but did not affect the sympathetic response. Pharmacological experiments with specific blockers to the VGCC subtypes showed that the Q-type VGCC is closely linked to the genesis of the parasympathetic response. We suggest that the anti-VGCC antibody from the LEMS patients specifically reduces the ACh release from the parasympathetic nerve by binding to the Q-type VGCC.
Between May 1990 and February 1995, 16 patients with adenocarcinoma or adenosquamous carcinoma of the uterine cervix were prescribed neoadjuvant chemotherapy consisting of cisplatin (50 mg/m2) on day 1, mitomycin C (10 mg/m2) on day 1, and etoposide (100 mg/m2) on days 1, 3, and 5 (MEP). In 2 patients stage was IB1, 5 were in stage IB2, 1 was in stage IIA, 5 were in stage IIB, 2 were in stage IIIB, and one was in stage IVB. A median of three courses of chemotherapy was given (range two to five). Of the 16 patients, 3 had a complete response and 5 had a partial response (response rate, 50%). Following termination of this chemotherapy, 12 patients with stage I or stage II carcinoma underwent radical hysterectomy. Three were given adjuvant radiotherapy because of positive pelvic nodes. One stage IIB patient, 1 stage IIIB patient and 1 stage IVB patient underwent standard radiotherapy and 1 stage IIIB patient underwent chemotherapy with another regimen because MEP therapy was without effect. Histopathological examinations revealed that changes as a result of the chemotherapy correlated well with clinical responses. Moderate or marked pathological changes occurred in 3 with a clinically complete response. The mean survival period of responders was 47.5 months while that of nonresponders was 28.3 months. Side effects of chemotherapy with MEP were within acceptable limits. The dose-limiting toxicity was myelosuppression and for only 1 patient the dose was reduced because of thrombocytopenia. Our preliminary study indicates that this chemotherapy regimen is effective for subjects with adenocarcinoma of the cervix. A prospective cooperative group trial on this regimen is ongoing.
There are no objection against that pulmonary vascular resistance index (PVRI) is one of the most important factors for completion of successful application of Fontan-type operation. However, calculated PVRI in single ventricle physiology in often unreliable because of difficulty in accurate measurement of pulmonary blood flow, especially in patients with decreased pulmonary blood flow. Although the role of bidirectional cavopulmonary shunt (BCPS) in such patients has been increasingly recognized, the impact of BCPS on PVRI has not been fully understood. Between November 1993 and November 1996, 24 patients, aged between 0.54 and 22.2 years, with a wide variety of cardiac malformations underwent BCPS, and were followed up for the mean of 15.1 months. There were four hospital deaths (16.7%) and three deaths in follow-up (12.5%). Serial catheterization revealed that significant increase in mean arterial oxygen saturation from 75.8% to 83.9% (p = 0.005), and decrease in mean Nakata's index from 433 to 311 (p < 0.0001). PVRI calculated by using formulas derived from Ohm's law before BCPS (Pulmonary flow was derived from Fick formula) was highly (greater than 10 u.m2) or moderately (between 4 and 10 u.m2) elevated in 6 and 7 patients, respectively. However, PVRI in these patients was normal after BCPS. Fourteen out of 24 patients underwent total cavopulmonary connection (TCPC) with 8 to 15 months of interval from BCPS, and have been currently surviving, and in NYHA functional class I or II, except 2 patients who underwent take-down. In conclusion, early and midterm outcome after staged operation appears to be excellent. BCPS is a good interim procedure, in part because one can more properly select patients undergoing Fontan operations from the PVRI point of view.
OBJECTIVE: To examine the critical point at which telomerase activation occurs in the course of cervical carcinogenesis. METHODS: Telomeric repeat assay protocol was used to measure telomerase activity in cell samples obtained from 155 Japanese women with various cervical conditions: normal cytology (n = 62), cervical intraepithelial neoplasia (CIN) (n = 63), and invasive squamous cell carcinoma (n = 30). RESULTS: Telomerase activity was detected in five (8%) women with normal cytology, in 26 (41%) patients with CIN (26% of patients with CIN I, 35% with CIN II, and 68% with CIN III), and in 29 (97%) patients with invasive carcinoma. Telomerase activation was significantly more frequent in CIN than in normal cervices (P < .001), and the positive rate in CIN III was significantly higher than that in CIN I (P < .01) and CIN II (P < .05). Furthermore, telomerase activation was significantly more frequent in invasive carcinoma than in CIN III (P < .01). CONCLUSION: Our findings suggest that telomerase activation is a relatively early event in cervical carcinogenesis and correlates well with grade of cervical lesion.
Two new potent serine protease inhibitors, cyclotheonamides E2 (3) and E3 (4), have been isolated from a marine sponge of the genus Theonella. Their structures were determined by interpretation of spectral data and chemical degradation studies. They are closely related to the previously reported cyclotheonamide E, from which they differ in the N-acyl group of the alanyl side chain. Cyclotheonamides E, E2, and E3 were more active against thrombin than against trypsin.
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We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis.
The standard telomeric repeat assay protocol (TRAP) was used to examine telomerase activity in 16 ovarian tumors, 16 cervical carcinomas, 4 uterine tumors, and 3 vaginal tumors. Telomerase activity was detected in 95% of these tumors, 88% of ovarian malignancies, and 100% of cervical, endometrial, and vaginal malignancies. In contrast, telomerase activity was not evident in normal tissues or in benign proliferative lesions, such as leiomyomas, condyloma acuminata, and simple endometrial hyperplasia. These results suggest that telomerase activation is associated with immortalization or malignant transformation of gynecologic tumors.
Telomerase activity is found in the majority of human cancers, but not in most normal tissues. It is generally accepted that there is a multistep process in human carcinogenesis. Studying the role of telomerase activation in this process may provide new information to further our understanding of the pathological process of clinical lesions. In the present study, telomerase activity was found in HPV-immortalized and cigarette smoke condensate (CSC)-transformed malignant cell lines established in a cervical carcinogenesis model and in cell lines derived from cervical intraepithelial neoplasias (CINs) and carcinomas. With exfoliated cell samples, telomerase activity was detected in 3 of 41 (7%) normal cervices, 10 of 25 (40%) CINs, and all 20 (100%) carcinomas. Telomerase activation was significantly higher in CINs than in normal cervices (chi2 = 7.42, P < 0.01) and was much higher in invasive carcinomas than in CINs (chi2 = 18.00, P < 0.005). Our observations suggest that telomerase activation is a relatively early-stage event in cervical carcinogenesis, and this activation is associated with the initiation and progression of cervical lesions. Detection of telomerase activity may serve as a tool for diagnosis and prognosis of cervical neoplasias.
The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.
The extracellular fluid pathway in the facial nerve and the diffusion of a tracer from the facial nerve to other cranial nerves was examined in the rabbit. Sodium fluorescein solution was injected into either the facial nerve fascicles or the epineurial connective tissue as a tracer at the stylomastoid foramen and then localized by fluorescence microscopy. In the facial nerve, fluorescence was observed in the endoneurium and external nerve sheath (epineurium and perineurium) through the geniculate ganglion following injection into the nerve fascicles. The vestibular, trigeminal, and glossopharyngeal ganglia also showed fluorescence on the injection side in ganglion cells and intercellular connective tissues. The results suggested that the endoneurial connective tissue constitutes a diffusion pathway inside the facial nerve fascicles and that the extracellular fluid pathway from the facial nerve to these cranial ganglia may be related to the neural spread of inflammation or neoplastic metastasis.
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There are few reports on the postoperative recurrence of bronchogenic cysts. We conducted a re-operation on a 57-year-old man with a bronchogenic cyst 15 years after an initial operation. His history showed an earlier operation for a bronchogenic cyst at the age of 42 at another hospital. 15 years after this initial operation, he suffered from common cold like symptoms, and was referred to our hospital, because of an abnormal shadow on his chest X-ray. A chest CT and MRI revealed an oval tumor just under the right intermediate bronchus. We suspected it was a recurrence of the bronchogenic cyst, and an operation was performed. The cyst was firmly adhered to the lung, and at the upper site of the cyst, a region adjacent to the intermediate bronchus was adhered to the bronchial wall. The histological findings were similar to those of 15 years previously. The cyst wall lined with pseudostratified columnar ciliary epithelium with muscular layer, which led to a diagnosis of a bronchogenic cyst. Congenital cysts, including bronchogenic cysts, are considered to originate in abnormal primordia. If there are remaining abnormal primordia, a recurrence of the disease can occur.
A number of epidemiological studies indicate that cigarette smokers are at increased risk of developing cervical cancer. However, convincing biological evidence is lacking. This report examines the biological and cellular role of human papillomavirus (HPV) type 16 and cigarette smoke in multistage cervical carcinogenesis. Two lines of HPV 16-immortalized human endocervical cells (HEN-16 and HEN-16-2) generated from primary cells (HEN) were treated with cigarette smoke condensate (CSC). CSC-treated, but not untreated, HEN-16 and HEN-16-2 formed tumors that were invasive squamous cell carcinomas in nude mice. The tumors were used to initiate 2 tumor lines of cells (HEN-16T and HEN-16-2T, respectively). Cells of both tumor lines, compared with HEN, HEN-16 and HEN-16-2, featured: (a) tumorigenicity, (b) distinct morphologies in monolayer and organotypic (raft) cultures, (c) faster growth in serum plus high calcium levels after immortalization and after transformation, (d) higher saturation density and (e) anchorage-independent growth. Our results provide unique direct in vitro evidence that cigarette smoke causes cancer in HPV-containing cervices.
Carcinogenesis originating from cervical cells has been recognized as a multistage process in which human papillomavirus (HPV) infection and cofactors, such as cigarette smoke, are frequently involved in the development of malignant cancer. However, the molecular mechanism underlying this process remains poorly understood. To identify the cellular genes involved in multistage cervical oncogenesis, we used the mRNA differential display method to analyze primary human endocervical cells (HEN), HPV 16-immortalized cells (HEN-16) and cigarette smoke condensate (CSC)-transformed cells (HEN-16T). Two cDNAs--PA4 and PA9--have been identified and isolated after comparing 8000 cDNAs from HEN, HEN-16 and HEN-16T. Northern blot analysis showed that PA4 was expressed 2- to 3-fold higher in HEN-16 and HEN-16T than in HEN, whereas PA9 was expressed uniquely in HEN. Moreover, the same patterns of PA4 and PA9 expression were found for a second line of HPV 16-immortalized endocervical cells and the corresponding transformed cells. An analysis of cDNA sequences showed that PA4 and PA9 had no homology to nucleotide sequences in Genbank. We suggest that immortalization, but not tumorigenesis, up-regulated a new oncogene, PA4, and down-regulated a new tumor suppressor gene, PA9. These results demonstrated the utility of the human endocervical cell model system and the mRNA differential display method for identifying the genes that may be involved in multistage cervical carcinogenesis.