Collagen abnormalities in the spinal cord from patients with amyotrophic lateral sclerosis

During the last 10 years, we have demonstrated morphological and biochemical abnormalities of skin extracellular matrices in amyotrophic lateral sclerosis (ALS). However, currently little is known concerning collagen of the spinal cord in ALS. We measured the amount of collagen and characterized collagen at light and electron microscopic levels in posterior funiculus, posterior half of lateral funiculus and anterior horn of cervical enlargement of the spinal cord obtained from ten patients with ALS, 11 patients with other neurologic diseases (control group A), and ten patients without neurologic ones (control group B). In posterior half of lateral funiculus and anterior horn, (1) by light microscopy, there was no significant difference in vessel wall area between ALS patients and control groups A and B; (2) ultrastructurally, collagen bundles were more fragmented and widely separated, and the fibrils were randomly oriented in the perivascular space of capillaries in ALS patients, which were not observed in any areas of control groups or in posterior funiculus of ALS patients; and (3) the collagen contents in ALS were significantly lower (P<0.001 and P<0.001, respectively) than those in control groups A and B. Fragmented and widely separated collagen bundles in the interstitial tissue surrounding capillaries and markedly decreased amount of collagen in posterior half of lateral funiculus and in anterior horn of ALS could be related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS, that is, selective neuronal vulnerability in ALS.     

Myc genes linked to viral promoters can collaborate with a transformation-deficient simian virus 40 early gene mutant to transform established rat cells

Cotransfection of a transformation-deficient and immortalization-positive SV40 early gene mutant with various constructs of viral promoter-linked myc genes induced dense foci in 3Y1 cells, an established cell line derived from rat embryo fibroblasts, whereas transfections with either species alone did not. Four cell clones were isolated from colonies grown in soft agar medium, and found to contain at least a single copy of both DNA species and to express both sequences at relatively high levels. These results indicate that the two immortalizing genes used in the present study collaborate to transform 3Y1 cells.     

A Case of Glucagonoma with Liver Metastasis: Complete Response to Dacarbazine

Abstract: We report a case of glucagonoma syndrome with liver metastasis, who responded completely to dacarbazine chemotherapy. A 77-year-old woman complained of itching skin eruptions (diagnosed as necrolytic migratory erythema) and weight loss. She was found to have glucose intolerance, anemia, hypoproteinemia and hyperglucagonemia. Abdominal CT and celiac arteriography showed a hypervascular tumor in the pancreatic tail and a metastatic tumor in the left hepatic lobe. Immunohistochemical examination of the metastatic liver tumor obtained by laparoscopic biopsy revealed the tumor cells to be positive for glucagon. The patient was treated with 20 courses of 300 mg/day intravenous dacarbazine for 5 consecutive days followed by a 4 week drug-free interval. No major side effects were noted. Treatment resulted in disappearance of the skin lesions and correction of anemia, glucose intolerance, hypoproteinemia and hyperglucagonemia. Follow-up abdominal CT showed complete resolution of both the primary pancreatic tumor and the metastatic liver tumor. We suggest that dacarbazine be considered as the treatment of choice for metastatic glucagonoma.     

Metastatic Behavior and Tumorigenicity of a Human Melanoma Cell Line (MM-RU) after Injection into Nude Mice

The ability of the human amelanotic melanoma cell line MM-RU to produce experimental metastases and to grow tumors at subcutaneous inoculation sites in 4-week-old nude mice was examined. After i.v. inoculation of 10⁶ cells, all injected mice (n=21) developed consistent numbers of metastatic pulmonary colonies within 32 days. The coefficients of variation for the number of colonies were between 17%–23% in three independent experiments. Survival time after i.v. inoculation was 63 ± 7 days (mean ± SD) (n=20). Within 20 days, subcutaneous inoculation of 5 × 10⁶ cells resulted in tumor growths of 13 ± 3 mm (mean ± SD) at the inoculation sites in all nude mice (n=12). The MM-RU cell line seems to be a simple, fast vehicle for testing the effect of melanoma growth modulators on experimental pulmonary metastases as well as on subcutaneously growing melanoma.     

In vivo activity on murine tumors of a novel antitumor compound,N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190)

Summary A novel antitumor compound, N--dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]-phenazine-6-carboxamide sodium salt (NC-190) was evaluated for its antitumor activity in experimental murine tumor systems. In the initial studies with P388 leukemia (i.p.-i.p.), NC-190 led to an increase of >200% in life span (ILS), and 75% of the mice were alive on day 30, when the optimal dose (50 mg/kg, days 1–5) was given. Additionally, the compound had significant activities against i.p. inoculated mouse L1210 leukemia, B16 melanoma, M5076 reticulum cell sarcoma, sarcoma 180, mouse hepatoma MH134, and rat Yoshida sarcoma and Yoshida ascites hepatoma AH130. The optimal dose resulted in a >280% ILS with a 30-day survival of 50% in mice with L1210 leukemia (100 mg/kg, days 1–5), a 156% ILS in mice with B16 melanoma (50 mg/kg, days 1–5), a 98% ILS with a 90-day survival of 25% in mice with M5076 reticulum cell sarcoma (25 mg/kg, days 1, 5, 9, and 13), a >300% ILS with a 60-day survival of 50% in mice with sarcoma 180 (50 mg/kg, days 3–10), a 148% ILS with a 60-day survival of 25% in mice with MH134 (25 mg/kg, days 1–5), a 129% ILS with a 60-day survival of 12.5% in rats with Yoshida sarcoma (12.5 mg/kg, day 3–10), and a >161% ILS with a 60-day survival of 50% in rats with AH130 (6.3 mg/kg, days 3–10). In the experiments with s.c. inoculated tumors, NC-190 not only inhibited tumor growth, but also increased the life span of mice with Lewis lung carcinoma or B16 melanoma. The 60-day survivors accounted for 60% and 30% in mice with Lewis lung carcinoma and B16 melanoma, respectively. The compound significantly inhibited the spontaneous lung metastasis of Lewis lung carcinoma by more than 90% when eight daily i.v. injections were given. NC-190 was active by the i.p., s.c., and i.v. routes. Five consecutive daily i.p. doses (days 1–5) were more effective than a single dose (day 1), two doses (days 1 and 5), or three doses (days 1, 5, and 9). NC-190 warrants further study as a potential antineoplastic agent against human neoplasms, as it has a broad spectrum of antitumor activity and inhibits metastasis.Abbreviations ILS increase in life span - MST median survival time - MMC mitomycin C - ADM adriamycin - CPA cyclophosphamide - 5-FU 5-fluorouracil     

Recent Trends in the Treatment and Prognosis of Adult Leukemia with Characteristics of Patients in Japan: Transition during the Fifteen Years from 1971 to 1985

Patients with acute (2,569) and chronic (957) leukemia diagnosedat 19 institutes took part in the study on the "MultidisciplinaryTreatment of Leukemia" between 1971 and 1985 and were investigatedretrospectively. By dividing the 15 years into three five-yearperiods, we were able to compare patient ratios in the differentperiods. The proportions of acute to chronic leukemia casesshowed no obvious change; however, the proportions of casesdiagnosed as acute lymphocytic leukemia in acute leukemia showeda significant increase. The main chemotherapeutic drugs usedduring the three time periods were cytarabine or its analogues,the anthracyclines, 6-mercaputopurine and prednisolone, againstacute myelogenous leukemia, and the vinca alkaloids, prednisoloneand the anthracyclines against acute lymphocytic leukemia. Therate of complete remission from acute myelogenous leukemia mademarked progress, from 45.1% during 1971–1975 to 62.3%during 1981–1985, but that of acute lymphocytic leukemiashowed no significant progress, being 65% during 1971–1975and 69.7% during 1981–1985. The durations of remission,however, and the survival times for patients with acute lymphocyticleukemia, as well as for those with acute myelogenous leukemia,became significantly longer over the three periods. Median survivaltimes from chronic myelocytic leukemia were 37–40 mo inall three periods, showing no progress. There was a better prognosisin cases of chronic myelocytic leukemia with, than without,Philadelphia chromosome. Except for a low incidence of chroniclymphocytic leukemia in Japan, adult leukemia patients' characteristicsand prognoses seem to be almost the same in Japan as in theU.S.A. and Europe.     

Human serum albumin incorporating synthetic heme: red blood cell substitute without hypertension by nitric oxide scavenging

The administration of extracellular, hemoglobin-based oxygen carriers often elicits an acute increase in blood pressure by vasoconstriction. This side effect is now recognized to be due to the depletion of nitric oxide (endothelial-derived relaxing factor) by the extravasuated hemoglobins. We have recently found that the administration of a recombinant human serum albumin (rHSA)-based oxygen carrier involving synthetic tetraphenyporphinatoiron(II) derivative (FeP) (rHSA-FeP) does not induce such hypertensive action, because of its low permeability through the vascular endothelium. The heart rate responses after the rHSA-FeP injection were also negligibly small. Visualization of the intestinal microcirculatory changes clearly revealed the widths of the venule and arteriole to be fairly constant. The entirely synthetic rHSA-FeP becomes a promising material as a new type of red blood cell substitute.     

Compatibility in vitro of albumin-heme (O(2) carrier) with blood cell components

Recombinant human serum albumin including 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivaloylamino)phenylporphinatoiron(II) (albumin-heme; rHSA-FeP) is a synthetic hemoprotein that has sufficient capability to reversibly bind and release O(2) under physiological conditions (pH 7.3, 37 degrees C) similar to hemoglobin and myoglobin. In order to use this albumin-based O(2) carrier as a new class of red blood cell substitutes, its compatibility with blood cell components carefully was investigated in vitro. After the addition of the rHSA-FeP solution into whole blood at 10, 20, and 44 vol %, the FeP concentration in the plasma phase remained constant for 6 h at 37 degrees C in each group, and no significant time dependence was observed in the numbers of red blood cells, white blood cells, or platelets. The microscopic observations clearly showed that the shapes of the red blood cells had not been deformed during the measurement period. With respect to the blood coagulation parameters (prothrombin time and activated partial thromboplastin time), the coexistence of rHSA-FeP had only a negligibly small influence. Also the blood compatibility under dynamic flow conditions was evaluated using a microchannel array flow analyzer. All these results suggest that the albumin-heme has no effect on the morphology of blood cell components in vitro.     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Effects of recombinant human endostatin on a human neuroblastoma xenograft

New antitumor agents must be added to the current neuroblastoma treatment regimens to improve the clinical results. We investigated whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against human neuroblastoma in the human neuroblastoma xenograft model designated TNB9. When tumors on the back of nude mice grew to a weight of 90-95 mg, rhEndostatin 10 mg/kg/day was administered subcutaneously every day for 10 consecutive days. Mean relative tumor weight in mice administered rhEndostatin (n=5) was significantly less than that in controls (n=12) on days 2, 4, and 6 after the start of administration (p<0.01 on day 2, p<0.05 on days 4 and 6), and regression of tumor growth (TRW<1.0) was marked on day 2. The maximum inhibition rate (MIR) by rhEndostatin was 46.4%, indicating inefficacy, but it may not be appropriate to apply Battelle Columbus Laboratories criteria to this experimental model because rhEndostatin is a protein. After day 8, tumors in the experimental group increased in weight and were not statistically significantly different from those in controls. Recombinant human endostatin was used in tumors in the arterial system of the mouse in this experiment because eventually rhEndostatin, not recombinant mouse endostatin, may be used to treat advanced neuroblastoma in the clinical setting. The results show that there is little cross-reactivity of rhEndostatin with the human and mouse models and indicate that rhEndostatin could become an effective agent for the treatment of human neuroblastoma.