Involvement of FGF-2 in the action of Emdogain on normal human osteoblastic activity.

OBJECTIVE: The present study was designed to evaluate the pharmacological characteristics of Emdogain (EMD) on cell growth and cell activity in human osteoblasts. METHODS: Cell proliferation as well as several gene and protein expressions were examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) procedures in human osteoblastic cells (SaM-1) treated with EMD (30 microg ml(-1)). RESULTS: Treatment of osteoblasts with EMD significantly stimulated cell proliferation and fibroblast growth factor (FGF)-2 expression but decreased alkaline phosphatase expression. In addition, increases in cyclooxygenase (COX)-2 expression and decreases in matrix metalloproteinases (MMP)-1 expression were observed in osteoblasts treated with EMD. The effects of EMD on FGF-2 and MMP-1 expressions were not observed in osteoblasts treated with NS-398, an inhibitor of COX-2. The decrease in MMP-1 mRNA by EMD was prevented by treatment with antisense oligodeoxynucleotide (AS-ODN) for FGF-2. CONCLUSION: Emdogain showing both stimulation of cell proliferation and inhibition of cell differentiation has been shown to increase FGF-2 expression in the mediation of prostaglandin E2 and to decrease MMP-1 mRNA expression through the activation of FGF-2. FGF-2 may underlie in the action of EMD on osteoblasts during periodontal regeneration.

Analysis of open reading frame 5 in Japanese porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism.

For the epidemiological survey of porcine reproductive and respiratory syndrome virus (PRRSV) isolated in Japan, the open reading frame (ORF) 5 gene of 37 field isolates in Chiba prefecture from 1991 to 1999 were analyzed by restriction fragment length polymorphism (RFLP). Reverse tanscription-polymerase chain reaction (RT-PCR) amplifying the ORF 5 gene detected 35 field isolates except for 2 isolates. RFLP analysis with MluI, HincII, SacII, HaeIII and MspI demonstrated that 35 field isolates were divided into 14 distinct codes and 34 isolates were distinguished from VR2332-derived modified live PRRSV vaccine, indicating the existence of genetic diversity in PRRSV field isolates in Japan. Only one strain 98-4A had an RFLP pattern identical to the vaccine strain. Nine out of the 35 field isolates (25.7%) had the RFLP code 1-3-2-1-1 and these patterns occurred throughout the examination period. This suggests that these isolates are the prevailing strain of PRRS in Chiba prefecture. RFLP with 5 enzymes demonstrated that various strains existed in the same year, and that there were different codes on the same farm according to the year. These observations indicate that there is a genetic variation among field isolates in very limited regions and some viruses possess the ability to cause their own genomic substitutions within a herd in a short time.

Biosynthesis of vitamin B(6) in rhizobium.

The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates. Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde.

Production of Vitamin B6 in Rhizobium.

The production of vitamin B6 was studied in about 1,590 bacterial isolates from soil, and an isolate, 28-21, identified as Rhizobium leguminosarum was obtained as a vitamin B6 high producer. Then, the production of vitamin B6 by commercially available Rhizobium strains was examined, and many of the tested strains excreted large amounts of vitamin B6 into the culture broth. The best producer of vitamin B6 was R. meliloti IFO 14782, which produced 51 mg per liter. Media study for the vitamin B6 production was done with R. meliloti IFO 14782; the strain was able to excrete 84 mg of vitamin B6 per liter, 79 mg per liter of which was pyridoxol.

Expression of vascular endothelial growth factor and p53 protein in association with neovascularization in human malignant gliomas.

We examined the expression of vascular endothelial growth factor (VEGF) protein, p53 protein, and the MIB-1 index in 43 patients with malignant gliomas in relation to tumor vascularization by an immunohistochemical method. Factor VIII-related antibody was employed for the evaluation of the vascularity and endothelial proliferation. Of the 42 cases of malignant gliomas, 36 (86%) demonstrated immunoreactivity for VEGF in their tumor cells, whereas 22 (52%) had VEGF in their endothelial cells. There was a tendency for the vascularity to be correlated with the immunoreactivity for VEGF (coefficient, 0.340). In addition, a marked increase in endothelial proliferation was evident in cases showing moderate to strong positivity for VEGF as compared with the others. Immunoreactivity for VEGF was found mostly in the malignant gliomas without p53 overexpression and/or with p53 overexpression. However, statistical analysis revealed a correlation between the grade of p53 overexpression and the grade of VEGF expression (coefficient, 0.507), but not between the VEGF and MIB-1 index in our series. There was a tendency for the MIB-1 indices to increase in correlation with increasing vascularity.

Ruptured aneurysm at the origin of duplication of the middle cerebral artery--case report.

A 28-year-old male presented with a rare case of an aneurysm at the origin of duplication of the middle cerebral artery manifesting as subarachnoid hemorrhage. Preoperative angiography revealed duplication of the right middle cerebral artery and an aneurysm at its origin, which was successfully clipped. He was discharged with no neurological deficits. Congenital factors may be more important in the etiology of aneurysms associated with this anomaly.

Intra-arterial ACNU chemotherapy employing 20% mannitol osmotic blood-brain barrier disruption for malignant brain tumors.

The clinical effects and problems of intra-arterial water-soluble antitumor nitrosourea (ACNU) therapy following osmotic blood-brain barrier modification are discussed. Twenty-one patients with malignant brain tumors were divided into two groups. Group 1 consisted of 16 patients treated by operation, irradiation, and two or more courses of intracarotid infusion of ACNU 100 mg/body (1.7-2.2 mg/kg) following 20% mannitol 200 ml (1.3-1.6 ml/sec) (7 grade 4 astrocytomas, 5 grade 3 astrocytomas, and 4 others). Group 2 consisted of five patients treated by operation, irradiation, and repeated intracarotid infusion of ACNU 100 mg/body alone (grade 4 astrocytoma). The 2-year survival rate in Group 1 was 79% (11 of 14 cases followed up for longer than 2 years) and the 3-year survival rate was 67%. Five of seven grade 4 astrocytoma patients (71%) in Group 1 survived for more than 1 year 6 months, whereas four of five grade 4 astrocytoma in Group 2 died within 1 year 6 months. The measurement of the ACNU concentration in tumor tissues and blood in 11 brain tumors, after intracarotid infusion of ACNU with blood-brain barrier disruption, showed peak values in the tumor tissues of 3.02-32.53 micrograms/gm (mean, 9.67 micrograms/gm), about three to five times as high as that in blood in most cases. This method used in Group 1 appears to be relatively safe without permanent neurological deficits and offers a potential therapeutic effect when used in combination with appropriate premedication in suitable patients.

[MCNU delivery into malignant brain tumor and normal brain tissue by intravenous or intraarterial infusion].

In 13 Fischer 344 rats transplanted intracerebrally with 9 L gliosarcoma, 13 normal Fischer 344 rats and 4 clinical cases of malignant glioma, a new water-soluble nitrosourea (MCNU) was given and the concentration was measured in blood, tumor tissues, normal brains around the tumors and normal hemispheres by intravenous or intraarterial infusion of MCNU. At 5 min. after administration of MCNU 20 mg/kg (4-5 mg/body) in 9 L gliosarcoma bearing Fischer rats, mean MCNU concentration in the blood was not different between 20 micrograms/ml intravenous and 23 micrograms/ml intraarterial administrations whereas that in the tumor tissues by intracarotid infusion of MCNU was 40 +/- 14.4 micrograms/g which was about two times as much as 22.9 +/- 8.13 micrograms/g by intravenous infusion of MCNU. Mean MCNU concentration of normal brains around tumor tissues was 2.49 micrograms/g in intravenous and 8.95 micrograms/g in intracarotid infusion. MCNU concentration of tumor tissues in 4 cases of malignant gliomas was higher by intracarotid administration than by intravenous administration compared to that in the blood. Maximum tumor/blood ratio of MCNU was 1.94 in intracarotid administration for the malignant glioma. It is suggested that intraarterial administration was more useful than intravenous infusion as an administration route for malignant brain tumors.

[Diagnostic value of neuron specific enolase (NSE) in the cerebrospinal fluid of malignant brain tumor with intrathecal metastasis].

Neuron specific enolase (NSE) in the cerebrospinal fluid (CSF) and serum of 54 cases of brain tumors, in the tumor tissues of 10 brain tumors and in the cyst fluids of 7 brain tumors was measured by radioimmunoassay with NSE measurement kit (Eiken Chemistry co.) NSE values in the cerebrospinal fluid of 35 malignant brain tumors showed abnormal increase higher than 10 ng/ml in 17 cases (about 50%) of them, whereas all of benign cases were lower than 10 ng/ml. The means and standard deviations of NSE in the cerebrospinal fluid of malignant tumors were 20.63 +/- 20.78 ng/ml in the astrocytoma grade 3 and 4, 19.73 +/- 15.5 ng/ml in the medulloblastoma and 12.4 +/- 8.9 ng/ml in the germ cell tumor. NSE values in the CSF of 12 brain tumors with intrathecal metastasis were 28.0 +/- 18.9 ng/ml (mean +/- SD) showing about three times as much as those without intrathecal metastasis. There was significant difference between these groups (p less than 0.01). All cyst fluids including in 7 cases of brain tumors demonstrated high values of NSE even if they were benign tumor and their NSE values in CSF were normal. NSE values of brain tumor tissues had no correlation with malignancy, but were higher in the periphery of the tumors than in the center. NSE values of CSF in brain tumors might be changed in relation with the improvement or deterioration on clinical state and CT. It might be suggested that the measurement of NSE in the CSF had significance as a monitor of therapeutic efficacy and prognosis for the brain tumors.

[Therapeutic efficacy of intracarotid infusion of 20% mannitol with ACNU in Fischer rats with intracerebrally implanted 9L gliosarcoma].

The purpose of this study was to investigate the therapeutic effect of intracarotid infusion of 20% mannitol with ACNU chemotherapy in Fischer 344 rats with intracerebrally implanted 9L gliosarcoma, compared with giving them only ACNU intraperitoneally. Thirty 9L gliosarcoma bearing Fischer 344 rats were evaluated in the following 3 groups. Group I: control (no treatment); group II: treated by ACNU 20 mg/kg intraperitoneally on the 7th day after implantation of 9L gliosarcoma cells; group III: treated by intracarotid infusion of 20% mannitol 3.1 ml/min. and with the same dose of ACNU as in group II. Mean survival time after the inoculation of tumor cells into the brain was 15.1 days (group I), 21.8 days (group II) and 27.9 days (group III). In group II all tumor-bearing Fischer rats died within 24 days after inoculation of tumor cells, whereas in group III 5 out of 6 rats survived more than 25 days after them. Group III evidenced necrosis and degenerative findings with vacuole and microcyst without vascular proliferation in tumor tissues more than group II on the 7th day after ACNU treatment for the experimental brain tumor. On the 7th day after ACNU treatment of each group, BrdU labeling index was calculated in order to evaluate tumor proliferation. The Mean BrdU labeling index in tumor cells of group III demonstrated 7.5%, against 14.5% (about one half) in group II. From our experimental study of survival time and BrdU labeling index, it is suggested that osmotic blood brain barrier disruption chemotherapy by intracarotid infusion of 20% mannitol and ACNU was more effective than simple treatment by intraperitoneal injection of ACNU in the Fischer rats with intracerebrally implanted 9L gliosarcoma.

[The effect on the tumor vessel permeability by hyperosmotic blood brain barrier disruption].

The limited effect of chemotherapy on malignant brain tumors has been related to tumor cell insensitivity to the drugs and to their ineffectual delivery to the tumor. The studies by Groothuis and Neuwelt et al have shown that the studies of tumor vessel permeability show considerable variability in different areas of tumors and between different tumor models. The present studies used the 9L gliosarcoma model in Fischer 344 rats to evaluate the increase of tumor vessel permeability by osmotic BBB opening on drug delivery to the tumor, brain adjacent to tumor (BAT), and brain distant to tumor using cis-diamminedichloroplatinum (CDDP) as a chemotherapeutic agent which was water soluble and rarely permeable to BBB. In addition the difference of delivery to normal brain and tumor tissue were studied on intravenous or intracarotid administration of cisplatin with or without intracarotid infusion of graded (20%, 25%) hyperosmolar mannitol. Evans blue, which binds to plasma albumin, was used to provide a visual marker of BBB opening. 20% or 25% hyperosmolar mannitol infusion to right internal carotid artery for BBB disruption was done at 0.12 ml/sec for 30 sec with controlled respiration after temporally clipping of right common carotid artery. Then cis-diamminedichloroplatinum (CDDP) was infused at 0.5 mg/ml/100 gr (body weight). In control studies isotonic saline instead of mannitol was infused to intracarotid artery at identical rate and volume. In 14 9L gliosarcoma bearing rats and 3 normal rats cis-diamminedichloroplatinum (CDDP) delivery to tumor and normal brain.(ABSTRACT TRUNCATED AT 250 WORDS)

New anthracycline antibiotics, auramycins and sulfurmycins. II. Isolation and characterization of 10 minor components (C approximately G).

Following the discovery of new anthracycline antibiotics, auramycins A and B and sulfurmycins A and B, we found 10 minor analogues of auramycins and sulfurmycins, C, D, E, F and G, from the culture broth of a mutant strain of Streptomyces galilaeus OBB-111 and prepared 2 analogues as the chemical derivatives from auramycin G and sulfurmycin G. All analogues have a sugar moiety at C-7 position of the aglycones. These analogues exhibit activities against Gram-positive bacteria and P388 leukemia.