Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts.

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

Secretion of bioactive interleukin-6 and tumor necrosis factor-alpha proteins from primary cultured human fetal membrane chorion cells infected with influenza virus.

Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection.

Nucleotide excision repair of melphalan monoadducts.

The nucleotide-excision repair (NER) system removes bulky DNA adducts and is thought to be involved in resistance to chemotherapeutic drugs, which act by damaging DNA. In this study, we have investigated the ability of the NER system to recognize and excise melphalan monoadducts from a 140-mer DNA substrate. We show that rodent and human cell-free extracts (CFEs) excise 26-29-nt-long oligomers from a synthetic 140-mer containing centrally located melphalan adducts. CFEs from cell lines with mutations in xeroderma pigmentosum group F or G genes did not excise these alkylated oligomers; however, mixing the two CFEs restored excision activity to the level found with wild-type CFEs. These results demonstrate the ability of the NER system to excise melphalan monoadducts, and are consistent with the hypothesis that NER may be involved in resistance to melphalan chemotherapy.

Spectrum of N4-aminocytidine mutagenesis.

N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.

Presence of Vicia graminea lectin- and Vicia unijuga lectin-binding (Vgu) glycoproteins in human fetal membranes and some of their biochemical properties.

We have previously reported that Vicia graminea lectin (VGA)- and Vicia unijuga lectin (VUA)-binding glycoproteins (Vgu glycoproteins), malignant tumor-associated antigens, exist in human meconium and amniotic fluid. To examine the origin of Vgu glycoprotein, their presence, some of their chemical and serological properties and their biosynthesis in the human fetal membrane, amnion and chorion laeve and accompanying membrane cells were examined. Perchloric acid-soluble fractions were prepared from human amnion and chorion laeve, after which VUA-binding components (Vgu glycoproteins) were separated by HPLC and affinity chromatography using immobilized VUA. Biosynthesis of the antigens in primary cultured cells prepared from the amnion and chorion laeve were examined by pulse-labeling and immunoprecipitation using immobilized VUA and compared with those in cultured human cancer cells. The results indicated that the serological properties of VUA-binding components in fetal membranes were similar to those of meconium and amniotic fluid, that many molecular species of VUA-binding components were synthesized in amnion and chorion laeve cells and that about 40-50% of antigens synthesized are secreted from cells while antigens synthesized in cultured cancer cells human were hardly secreted with more than 95% of the antigens remaining in the cells. From these results, we concluded that a large part of Vgu glycoproteins found in amniotic fluid is synthesized in cells of the amnion and chorion laeve and secreted into the fluid, and that Vgu glycoproteins synthesized in cancer cells were not secreted, rather they were retained in the cells.

Exercise capacity of thoracotomy patients in the early postoperative period.

OBJECTIVE: We investigated the mechanism involved with the initial drop and subsequent recovery of exercise capacity in the early postoperative period of thoracotomy patients. METHODS: Sixteen patients (13 who had undergone lobectomy, 3 who had undergone pneumonectomy) underwent a routine pulmonary function test (PFT) and a cardiopulmonary exercise test preoperatively, within 14 postoperative days (POD; post-1; mean +/- SD, 9 +/- 2 POD), and after 14 POD (post-2; mean, 26 +/- 12 POD). RESULTS: After surgery on post-1, PFT results of FVC, FEV(1), and maximum ventilatory volume (MVV) significantly decreased. Oxygen uptake (VO(2)) at a venous blood lactate level of 2.2 mmol/L (La-2. 2), which was adopted as the empirical anaerobic threshold, and maximum V O(2) (VO(2)max) decreased significantly to 88.2 +/- 7.9% and 73.1 +/- 15.4% of the preoperative values, respectively. La-2.2 min ventilation (VE)/ MVV and maximum VEmax)/MVV increased significantly from 0.36 +/- 0.08 to 0. 66 +/- 0.20 and from 0.58 +/- 0.14 to 0.80 +/- 0.09, respectively. On post-2, though La-2.2 VO(2) did not change, VO(2)max improved significantly to 81.5 +/- 19.7% of the preoperative values, in association with significant increases in maximal tidal volume and VEmax, which were produced by significant increases in the PFT results. La-2.2 VE/MVV also decreased significantly to 0.49 +/- 0.13, which indicated a sufficient recovery of respiratory reserve at submaximal exercise. CONCLUSIONS: The initial drop of exercise capacity after lung resection seems to be derived from both circulatory and ventilatory limitations. Further, the subsequent recovery within 1 month seems to be produced by an improvement in ventilatory limitation, which was caused by the surgical injury to the chest wall.

Correlation between quantitative antibody titers of sperm immobilizing antibodies and pregnancy rates by treatments.

Immunological infertility in women who possessed sperm immobilizing (SI) antibodies made it very difficult to conceive using the usual treatments. We examined SI antibodies by the quantitative Sperm Immobilization Test and found the antibody titers (50% sperm immobilization unit: SI50 unit) associated with pregnancy rates. Patients with high SI50 titers (greater than 10 units) did not conceive by ordinary or repeated artificial inseminations with husband's semen (AIH) except when treated with in vitro fertilization (IVF) and embryo replacement. Patients with relatively low SI50 titers (less than 10 units) could conceive either by repeated or ordinary AIH, though the success rates were lower than by IVF-embryo replacement. It is important to assess the SI50 titers by the quantitative method to select treatments for infertile women with SI antibodies. In follow-up studies of the patients who conceived successfully, it was found that SI50 titers tended to decline as pregnancy proceeded.

Enhancement of the response to levodopa therapy after intrastriatal transplantation of autologous sympathetic neurons in patients with Parkinson disease.

OBJECT: There is growing evidence to indicate that tissue transplantation can potentially be a restorative neurosurgical treatment for patients with Parkinson disease (PD). In this study the authors investigated the clinical effect of unilateral intrastriatal grafting of autologous sympathetic neurons in patients with PD. METHODS: Four patients with PD who had been observed for 1 year after graft placement of autologous sympathetic neurons were selected for an analysis of the effect of that procedure. Sympathetic ganglion tissue was endoscopically excised from the thoracic sympathetic trunk and grafted into the unilateral caudate head and putamen of the PD patients. No changes were made in the patients' preoperative regimens of antiparkinsonian medications, and clinical evaluations were made principally according to those established by the Core Assessment Program for Intracerebral Transplantation Committee. Whereas the sympathetic neuron grafts failed to affect clinical scores reflecting the patients' motor performance, which was evaluated during either the "on" or "off' phases, the grafts significantly increased the duration of the levodopa-induced on period with consequent reduction in the percentage of time spent in the off phase. This beneficial effect may be explained by the results of the present in vitro experiment, which show that human sympathetic neurons have the ability to convert exogenous levodopa to dopamine and to store this synthesized dopamine. CONCLUSIONS: Sympathetic neuron autografts were found to improve performance status in patients with PD by reducing the time spent in the off phase. This clearly indicates that sympathetic ganglion tissue, the use of which involves few ethical issues, can be an efficacious donor source in cell transplantation therapy for PD. Further studies are needed to determine whether the grafts may provide long-lasting clinical benefits.

Acquisition of multidrug resistance in recurrent breast cancer demonstrated by the histoculture drug response assay.

Recurrent breast cancer has a very poor response rate to chemotherapy. To understand the degree of acquisition of multidrug resistance in recurrent disease, 24 recurrent breast tumors and 127 primary tumors were evaluated and compared for chemosensitivity in the histoculture drug response assay (HDRA). The evaluation rate was 98.8%. The HDRA utilizes 3-dimensional culture of human tumors on collagen-gel rafts. Doxorubicin (DXR), 5-fluorouracil (5-FU) and mitomycin C (MMC) were tested as standard agents and cisplatin (CDDP) as a candidate agent on surgical specimen of breast cancer in the HDRA. In vitro drug exposure in the HDRA was for 7 days. At the end of the assay, tumor response was assessed by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The mean inhibition rates of primary tumors vs. recurrent tumors were 57.9% and 38.6% for DXR (p<0.0005); 59.9% and 42.8% for MMC (p<0.01); 49.0% and 33.4% for 5-FU (p<0.01); and 34.5% and 16.0% for CDDP (p<0.005), respectively. The recurrent cases were pretreated clinically with CAF (cyclophosphamide, DXR and 5-FU), CEF (cyclophosphamide, epirubicin and 5-FU) or CMF (cyclophosphamide, methotrexate and 5-FU). In the CAF and CEF group, the HDRA sensitivity to CDDP was significantly lower in recurrent disease (p<0.005) than that of primary breast cancer suggesting that one agent can induce resistance to another. This is further suggested by the fact that 64.7% of the recurrent cases were resistant to all 4 agents tested as opposed to 27% of the primary cases and that only 5.9% of the recurrent cases were sensitive to three or more agents as opposed to 18% of the primary cases. The correlation of the HDRA results to clinical outcome in the study was 80.0% with 15 cases evaluated consisting of 5 true positives, 3 false positives, 7 true negatives and no false negatives. Thus, the HDRA gives useful clinical information, in particular for the specific individualized treatment design necessary to overcome the multidrug resistance problem of recurrent breast cancer.

Disappearance curves for tumor markers after resection of intrathoracic malignancies.

Determination of the standard elimination kinetics of tumor markers will be helpful in the diagnosis of malignancies. We analyzed the disappearance curves for serum tumor marker levels after resection of intrathoracic malignancies. Serum levels of CEA, SLX, AFP, CA 19-9, SCC, TPA and CYFRA were measured several times after surgery in a total of 40 patients. To obtain precise biological half-lives, we applied non-linear least square analysis, taking into consideration the possibility of residual tumor cells. Disappearance curves were monophasic for CEA, SCC, TPA, CYFRA and SLX and biphasic for CA 19-9 and AFP. Temporary elevation of serum levels after surgery was observed for SCC, TPA and CYFRA. The average half-lives of CEA, SLX, SCC, TPA and CYFRA were 1.5 days, 2.7 days, 2.2 hours, 2.5 hours and 1.5 hours, respectively. The average half-life of CA 19-9 was 0.5 days in the first compartment and 4.3 days in the second compartment, while that of AFP was 1.0 days and 6.3 days, respectively. These values will be helpful in the interpretation of serum tumor marker levels after surgery.

Static torque-angle relation of human elbow joint estimated with artificial neural network technique.

Static relations between elbow joint angle and torque at constant muscle activity in normal volunteers were investigated with the aid of an artificial neural network technique. A subject sat on a chair and moved his upper- and forearm in a horizontal plane at the height of his shoulder. The subject was instructed to maintain the elbow joint at a pre-determined angle. The wrist was then pulled to extend the elbow joint by the gravitational force of a weight hanging from a pulley. Integrated electromyograms (IEMGs), elbow and shoulder joint angles and elbow joint torque were measured. Then the relation among IEMGs, joint angles and torque was modeled with the aid of the artificial neural network, where IEMGs and joint angles were the inputs and torque was the output. After back propagation learning, we presented various combinations of IEMGs, shoulder and elbow joint angles to the model and estimated the elbow joint torque to obtain the torque-angle relation for constant muscle activation. The elbow joint torque increased and then decreased with extension of the elbow joint. This suggests that if the forearm is displaced from an equilibrium point, the torque angle relation would not act like a simple spring. In a view of the musculoskeletal structure of the elbow joint, the relation between the elbow joint angle and the moment arm of the elbow flexor muscles seems to have a dominant effect on the torque-angle relation.

Nucleoside and nucleobase analog mutagens.

Compounds with structures close to those of normal nucleosides or nucleobases may be incorporated into cells and then become constituents of their DNA. Proliferation of such cells could yield mutants. In this article, the current status of studies on such nucleoside and nucleobase analogs is described. Base mispairing mechanisms for these analogs are discussed in light of recent biochemical and biophysical findings.

Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions.

N4-Aminocytidine is mutagenic in various organisms. In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA. To prove that the N4-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA. We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations. The M13mp2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies. Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present.

Effects of maternally administered immunoglobulin on platelet counts of neonates born to mothers with autoimmune thrombocytopenia: re-evaluation.

OBJECTIVE: Since immunoglobulin is transported across the placenta, maternal administration of it theoretically seems attractive as an antenatal treatment for a fetus. However, the effects of antenatally administered intravenous immunoglobulin (IVIG) on fetal platelet counts have been controversial. Our series of 11 cases of idiopathic thrombocytopenic purpura (ITP) and a review of previous reports are presented. A combined retrospective analysis to know whether maternal response to IVIG is associated with improvement of fetal thrombocytopenia was conducted. METHODS: IVIG was given to 11 steroid refractory pregnancies with ITP. Good maternal response to the therapy was defined as an increase in the platelet count to greater than 50 x 10(9)/L after completion of the IVIG infusion. Neonatal platelet counts of the umbilical cord were performed just after birth and followed-up for at least the first week of life. RESULTS: Seven of the 11 neonates had thrombocytopenia of less than 100 x 10(9)/L, and two of them had severe thrombocytopenia of less than 50 x 10(9)/L. Two out of two neonates born to mothers with a good response to IVIG were thrombocytopenic; whereas five out of nine neonates born to mothers with a poor response were thrombocytopenic. The combined retrospective analysis of our results and published reports have shown that fetal thrombocytopenia was not associated with the maternal response to IVIG but with the level of immunoglobulin G of neonates at birth. Passive thrombocytopenia was more frequently observed in neonates with normal levels of immunoglobulin G than those with elevated levels. CONCLUSION: This combined analysis confirmed the proposed hypothesis that the lack of effect of maternally administered IVIG on fetal platelet counts may mainly be attributed to the insufficient therapeutic level of transferred immunoglobulin G in the cord blood.

[Surgical treatment of lung cancer with chest wall invasion].

From January 1986 to May 1998, 45 lung cancer patients with chest wall invasion (P3) underwent resection (40 male, 5 female), (median age 63.2 yrs (30-79)). Histological types were squamous cell carcinoma in 20, adenocarcinoma in 14, large cell carcinoma in 7, adenosquamous cell carcinoma in 2, and unknown in 2. Operative methods of lung resection were total pneumonectomy in 2, bilobectomy in 3, lobectomy in 38, and partial lung resection in 2. Resection was regarded as complete in 35 and incomplete in 10 patients. Thirty one patients had negative lymph nodes (N0), 9 had peribronchial or hilar lymph node metastases (N1), and 5 had mediastinal lymph node metastases (N2). The extent of tumor invasion to chest wall was P3a (invasion within parietal pleura) in 11, P3b-c (invasion to intercostal muscle) in 16, P3d (invasion to rib) in 18, patients. 5-year survival rate was totally 19.7%. Cisplatin based chemotherapy and concurrent thoracic radiation following surgery (CCRT) was performed in latest nine P3d cases. Partial response was observed in 5 of 9 cases (response rate 56%) and viable tumor cell in the primary site was not seen histologically in 5 of 9 cases. Three year survival rate was 46.9% for CCRT(+) 11.1% for CCRT(-). Acturial 5-year survival rate in P3a-d was 19.76%. P3d cases had poor survival, but CCRT improved prognosis of P3d cases.

[Experience with radical operation for lower thoracic esophageal cancer through left diagonal thoraco-laparotomy and right posterolateral thoracotomy].

Two cases were reported in which subtotal thoracic esophagectomy, total gastrectomy, splenectomy and distal pancreatectomy was performed for the lower thoracic esophageal cancer through right diagonal thoraco-laparotomy and left posterolateral thoracotomy. Reconstruction was done with the intrathoracic esophagojejunostomy. Extubation could be done on the 1st postoperative day, and postoperative course was uneventful. It was thought that the approach with left diagonal thoraco-laparotomy and right posterolateral thoracotomy was useful for the easy and complete lymph node dissection from the middle mediastinum to the intra-abdominal cavity.

[Skin sensitization of costus root oil and peripheral lymphocyte count in gastric and colon cancers].

Costus root oil is a new primary sensitizer to examine the delayed, cell-mediated immune system. The peripheral blood lymphocyte count has been measured in 68 cases of carcinoma of the stomach and colon. The count has been correlated with the stage of tumor spread and the patient's delayed hypersensitivity response to costus root oil. A statistically significant correlation was found between the peripheral blood lymphocyte count and the response to costus root oil. Patients with low peripheral blood lymphocyte counts tended to have more advanced tumors and a poor response to costus root oil.

[Induction chemotherapy based on histoculture drug response assay for a patient with advanced thymic cancer--a case report].

Histoculture drug response assay (HDRA) was applied to a biopsy specimen of advanced thymic cancer from a 68 years-old male patient. HDRA revealed that the tumor was not sensitive to CDDP but highly sensitive to 5-FU, ADM and MMC, which were administered as induction chemotherapy. The tumor regressed to 14% of the pretreatment size and was able to be completely resected with right and left brachiocephalic veins, superior vena cava, pericardium, right phrenic nerve and a part of right lung. Histologically, only a few small cancer nests remained in the fibrous tissue. The patient is alive and disease free 32 months after surgery. This result suggests that HDRA is useful to select anticancer agents which are sensitive to a rare kind of carcinoma such as thymic cancer.

[A five-year-survival case in which complete response was recognized after combined chemotherapy using 5-fluorouracil, adriamycin and mitomycin C (FAM) for unresectable gastric cancer].

FAM (5-fluorouracil, adriamycin, mitomycin C) therapy was performed on a 65-year-old man with unresectable gastric cancer. Cancer cells have not been recognized by endoscopic biopsy after the patient's complete response. He is alive without metastasis of recurrence for five years.