Sequence-dependent Termination of Mammalian DNA Polymerase Reaction by a New Platinum Compound, (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) Monohydrate

We examined the mechanisms of the inhibition of DNA synthesis by a new platinum compound, (-)-( R )-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis- diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase α were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase β was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.     

Risk factors for invasive aspergillosis in living donor liver transplant recipients.

Invasive aspergillosis (IA) is a severe complication of liver transplantation. Risk factors for IA after deceased donor liver transplantation (DDLT) have been presented in several reports, but are not well established for living donor liver transplant recipients. Here, a retrospective case-control study was performed. Five cases with IA were investigated after living donor liver transplantation (LDLT) between January 1999 and December 2002 at Kyoto University Hospital. For comparison, living donor liver transplant recipients without IA were taken as controls. These patients had undergone LDLT 1 month before or after each IA case and had the same survival times as the latter. We evaluated the clinical and laboratory findings for both groups up until their demise. Patients with IA after LDLT had a very poor prognosis. By univariate analysis, risk factors for IA were preoperative intensive care unit stay (P = 0.02) and preoperative steroid administration (P = 0.02). Preoperative steroid administration for fulminant hepatitis possibly predisposed to the development of IA after LDLT.     

5-HT(2) receptor-mediated phosphoinositide hydrolysis in bovine ciliary epithelium.

The serotonin 2 (5-HT(2)) receptor antagonists, MCI-9042 (Anplag) and ketanserin, have been shown to lower intraocular pressure in rabbits (1) and humans (2). The mechanism of action of these drugs has not been determined, but it is hypothesized that 5-HT(2) receptors, and possibly alpha-adrenergic receptors, (3) may regulate in part aqueous humor production via an intracellular signal transduction pathway in the ciliary body. We therefore examined whether 5-HT(2) receptors were coupled to phosphoinositide hydrolysis in an organ culture system of isolated bovine ciliary epithelium. 5-HT stimulated [(3)H]inositol phosphates ([(3)H]InsPs) accumulation in a dose-dependent manner with a maximum increase approximately twice over the basal level. The mean EC(50) value was 1.1 microM, which was calculated from four dose-response curves. The 5-HT stimulated accumulation of [(3)H]InsPs was inhibited by spiperone (5-HT(2A/1A) and dopamine 2 (D(2)) antagonists), M-1 (a major metabolite of MCI-9042), ketanserin (5-HT(2A) antagonist), SB-206553 (5- HT(2B/2C) antagonist), and mesulergine (5-HT(2C) antagonist and D(2) agonist). It was not inhibited by chlorpromazine, which is a D(2) receptor antagonist. Accordingly, our study demonstrates that 5-HT(2) receptors are coupled to phospholipase C in bovine ciliary epithelium.     

Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized ldl receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis.

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.     

Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor).

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage. INTRODUCTION: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2. MATERIALS AND METHODS: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo. RESULTS: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro. CONCLUSION: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.     

Surgical Treatment of Intractable Seizures Due to Hypothalamic Hamartoma

Summary: A 6.5-year-old boy developed seizures at age 2.8 years consisting of episodes of unconsciousness and laughing attacks. By age 6 years, multiple seizure types, including generalized tonic-clonic (GTC), complex partial (CPS) and akinetic seizures, and drop attacks were occurring several times daily. EEG showed multifocal epileptic discharges. Antiepileptic drugs (AEDs) did not control the seizures. With progression of the epilepsy, cognitive deterioration developed. There were no manifestations of precocious puberty. Neuroimaging disclosed a suprasellar mass in continuity with the hypothalamus, and a diagnosis of hypothalamic hamartoma was made. After surgical resection of the hamartoma, the seizures were completely alleviated, and the epileptic EEG discharges disappeared. Improvement of mental function was also noted.     

Clinical usefulness of the WHO histological classification of thymoma.

PURPOSE: Rosai et al. published the World Health Organization (WHO) classification of thymic epithelial tumors in 1999, and its clinical usefulness seems to be established. It is our purpose to find the clinically relevant diagnostic points in the WHO Histological Classification of Thymoma. METHODS: Thymomas surgically removed from 100 consecutive patients at Juntendo University Hospital between October 1983 and February 2002 were classified according to the WHO histological classification. We assessed overall survival and recurrence-free rate calculated for each tumor type in the WHO classification compared with those of tumors classified by the Masaoka system. RESULTS: The thymic epithelial tumors in this series comprised 10 type A, 15 type AB, 18 type B1, 21 type B2, 33 type B3, and 3 type C tumors according to the WHO classification. Based on the Masaoka system, the disease was stage I in 53 patients, stage II in 30, stage III in 15, and stage IV in 2. The 15-year recurrence-free rate was 100% for type A, AB and B1, while the rates for types B2 and B3 were 66.7% and 54.5%, respectively. The 10-year recurrence-free rate was 66.7% for type C. The 15-year recurrence-free rate of the 64 patients with type A, AB, B1, and B2 thymomas was significantly higher from that of the 33 patients with type B3 thymoma (p=0.0026). CONCLUSION: When using the WHO classification, it is critical to distinguish type B3 thymoma from other tumor types.     

Germ cell tumor in the hypothalamo-neurohypophysial region: clinical features and treatment

Twenty-one patients with germ cell tumors (17 germinomas and 4 teratomas) involving the hypothalamic-neurohypophysial (HN) region were reviewed retrospectively. Eleven patients were males and 10 females, and their ages ranged from 7 to 45 years (average 18.5 years). Diabetes insipidus was the initial and the most prominent symptom in most germinomas; in teratomas the most prominent symptom was visual disturbance. Fifteen patients with germinomas were treated by radiotherapy, and 4 with teratomas were treated by surgical resection alone. Two recent germinoma patients with extensive CSF dissemination were treated with systemic chemotherapy consisting of anticancer platinum drugs and etoposide, which resulted in a complete disappearance of the tumors. Patients with germinoma treated after the introduction of CT scanning had a greatly improved mortality rate, and their actual survival rate was 87.5% over 10 years. On the basis of this review, the authors consider that diagnosis at an early stage of the disease and chemotherapy, which can be an effective therapeuric alternative to radiation therapy, may improve not only the mortality rate but also the quality of life of patients with HN germ cell tumors.     

Open reduction and varus-detorsion osteotomy with femoral shortening in treatment of congenital dislocation of the hip

In this study, we clinically and radiographically evaluated open reduction with shortening of the femur in children more than 1 year old with refractory congenital dislocation of the hip. In 19 children (aged 1–4 years), 22 joints were operated on. The patients were followed-up for an average of 8.7 years (range, 2–13 years). Functional results were satisfactory in all joints, and differences in limb length were not significant. Radiographically, good results (grades I and II) were obtained in 16 of the 22 joints, according to Severin's criteria. This surgical procedure may be indispensable for treating refractory congenital dislocation of the hip in children over 1 year old. Received for publication on May 2, 1997; accepted June 3, 1998     

The effect of a mixture of tegafur with uracil (UFT) on hepatocarcinogenesis in rats induced by 3'-methyl-4-dimethylaminoazobenzene. I. The influence on production of alpha-fetoprotein

We studied the effect of UFT (a mixture of Tegafur and uracil at a ratio of 1:4) on hepatocarcinogenesis and alpha-fetoprotein (AFP) production induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in rats. By feeding rats with 3'-MeDAB diet, serum AFP started to appear from week 2-3 and increased until week 5-7, then declined until week 10-11. This phenomenon of a transient appearance of serum AFP is called "primary reaction". After week 11 hepatoma started to develop accompanied with an enormous elevation of serum AFP level until animals die, generally before week 25. Male Donryu strain rats fed a diet containing 0.06% 3'-MeDAB for 10 weeks, and then fed the normal diet. UFT was given by a stomach tube once a day for five days a week. Blood was collected from the tail vein every other week and serum AFP was measured by Rocket electrophoresis technique. When the administration of UFT (10 mg or 20 mg/kg/day) was started simultaneously with feeding 3'-MeDAB diet, the primary reaction was somewhat suppressed, and after week 11 development of hepatoma and elevation of AFP were markedly inhibited. The administration of UFT of a high dose (40 mg/kg/day) caused the death of animals in 7 weeks probably because of its toxicity. When the administration of UFT (10 mg or 20 mg/kg/day) was started after finish of feeding 3'-MeDAB diet for 10 weeks, the drug was not effective. These results show that if the administration of UFT was commenced at the same time with feeding 3'-MeDAB diet, the hepatocarcinogenesis and AFP production are strongly inhibited.