Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C

Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.     

Enhancement of sensitivity to CDDP in human pharyngeal carcinoma cells by H-7, a protein kinase inhibitor

We investigated the synergetic effects of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) on cis-diamminedichloroplatinum(II) (CDDP) cytotoxicity to KB cells, a human pharyngeal carcinoma cell line, and explored the possible mechanism whereby it exerted these effects. In vitro treatment with H-7 markedly decreased protein kinase C (PKC) activity in KB cells and significantly sensitized the cells to CDDP. Treatment of KB cells with H-7 induced a dose-dependent depletion of intracellular glutathione (GSH) levels. These results suggest that the decreased GSH content induced by H-7 may be involved in sensitizing KB cells to CDDP.     

Coronary artery bypass grafting in two patients with single coronary artery

We experienced two patients with single coronary artery who underwent CABG using arterial grafts successfully. In two patients coronary angiography demonstrated a single coronary artery which was originated in left coronary sinus and was bifurcated to LAD and LCx, and then RCA branched off proximal LAD, passing in front of the right ventricular out flow tract (Sharbaugh Type L-IIa). To the first patient, a 52-year-old man who had angina on exertion due to long stenosis of RCA, CABG to RCA using RITA was carried out. To the second patient, a 57-year-old man who had inferior myocardial infarction due to 90% stenosis of proximal LAD, CABG to RCA using RITA and LAD using LITA was carried out. Single coronary artery without additional congenital cardiac anomalies may lead to myocardial ischemia, necessitating CABG as coronary reconstructions.     

IgA interaction with carboxy-terminal 43-kD fragment of fibronectin in IgA nephropathy

IgA deposition in the glomerular mesangial matrix is a prerequisite for the diagnosis of IgA nephropathy, and circulating IgA-containing complex has been implicated in this process. Since fibronectin is known to be involved in the assembly of extracellular matrix, this study was conducted to investigate whether fibronectin and its fragments are present in sera of patients and are capable of binding IgA1. Sera from patients with IgA nephropathy were purified by heparin-affinity chromatography, and column eluate were analyzed for the presence of fibronectin using Western blot and a set of anti-fibronectin monoclonal antibodies. Native fibronectin was digested with cathepsin D to obtain fragments similar to those of serum fibronectin. The capacity of fibronectin to bind IgA was examined with a mixture of purified IgA1 and cathepsin D-digested fibronectin fragments. A 43-kD carboxy-terminal fragment of fibronectin was detected in samples derived from sera of patients with IgA nephropathy but not in healthy control subjects. A similar-sized fragment was generated by cathepsin D digestion of the native molecule and was shown to bind to IgA1 in vitro. Since the carboxy-terminal domain is known to be critical in assembling exogenous fibronectin into the extracellular matrix, the affinity to IgA1 to a fragment found in patients may have pathogenic potential to mediate extracellular IgA deposition in IgA nephropathy.     

Apoptotic cascade of neurons in the subcortical sensory relay nuclei following the neonatal infraorbital nerve transection

A terminal transferase-mediated dUTP nick end labeling (TUNEL) method was utilized for detection of neuronal death in the subcortical relay nuclei of the trigeminosensory system following the infraorbital nerve transection in newborn rats. At 18-24 h after injury, numerous TUNEL-positive profiles were found within the ventroposteromedial thalamic nucleus (VPM) contralateral to the injury, whereas the VPM on the ipsilateral side and of the age-matched normal control contained only a few profiles per section. Electron microscopy revealed that the TUNEL-positive profiles were apoptotic neurons. The ventral part of the ipsilateral brainstem sensory trigeminal nuclear complex (the nucleus principalis, and the subnuclei oralis and interpolaris) exhibited statistically significant 65-70% increase in number of apoptotic neurons compared to the contralateral side. Taken together with our previous study [T. Sugimoto, C. Xiao, H. Ichikawa, Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism, Brain Res. 807 (1998) 147-154], the present results demonstrated a cascade of apoptosis in the primary, secondary and tertiary order sensory neurons along the neuroaxis.     

The inhibitory effect of recombinant human soluble thrombomodulin on initiation and extension of coagulation--a comparison with other anticoagulants

Recombinant human soluble thrombomodulin (rhsTM) was compared with various anticoagulants for in vitro anticoagulant effects on thrombin generation, clotting time, and thromboelastography. rhsTM as well as APC reduced the level of the peak of the thrombin generation curve, but we did not observe any time-delay to reach the peak. This effect of rhsTM was diminished in PC-deficient plasma and was closely associated with the inhibitory effect on prothrombinase and factor Va. On the other hand, hirudin and argatroban delayed the time to reach the level of the peak, without reducing it. rhsTM and other anticoagulants except for activated protein C (APC) were found to have concentration-dependent anticoagulant activity by conventional clotting tests. However, the concentration of rhsTM for clotting time was slightly affected by anti-protein C antibody. Moreover, the concentration of rhsTM required to inhibit thrombin activity directly was 50 times higher than that needed to inhibit thrombin generation. The effect of rhsTM on clot development was compared with that of other anticoagulants by thromboelastography; rhsTM reduced the growth of the clot but had little effect on the time to activate clotting, while the other anticoagulants had the opposite effect. This effect of rhsTM was completely abolished by the addition of anti-protein C or anti-protein S antibody. These findings suggest that rhsTM attenuates blood clotting by reducing the level of generated thrombin through protein C activation and subsequent factor Va inactivation and prothrombinase inhibition.     

RhoN, a novel small GTP-binding protein expressed predominantly in neurons and hepatic stellate cells

A cDNA encoding a novel member of the small molecular weight GTP-binding protein (small G-protein) superfamily was cloned from rat spinal cord. The deduced amino acid sequence was highly homologous with those of so-far-known Rho proteins. Rho proteins were reported to alter many important cellular functions including formation of both actin stress fibers and focal adhesions. RNA blot hybridization and in situ hybridization analyses indicated that the novel small G-protein is expressed specifically in neurons in the brain and spinal cord and also in hepatic stellate cells. Based on the sequence similarity and neuron-specific expression in the brain, this protein was named RhoN. Unlike classical Rho proteins, RhoN was not susceptible to the ADP-ribosylation reaction by C3 botulinum toxin. Accordingly, RhoN seemed to be specifically involved in neuronal and hepatic functions as a C3 toxin-insensitive member of the Rho subfamily. Then, a mouse genomic DNA segment containing the RhoN gene was cloned. The locus was mapped on the mouse chromosome 11C-D. The sequence data showed that the protein-coding sequence for RhoN is divided by 4 introns, and that the defined 5 exons may encode intramolecular domains serving for different functions.     

The expression of Ca2+/calmodulin-dependent protein kinase I in rat retina is regulated by light stimulation

Ca2+/calmodulin-dependent protein kinase I (CaM-kinase I) in rat retina was analyzed by immunohistochemical analysis, Western blot analysis and kinase activity assay. Western blot analysis revealed two immunoreactive bands similar to those detected in the brain. Developmental studies revealed that CaM-kinase I expression increased in accordance with postnatal development. Expression of CaM-kinase I in the retinas of rats raised in the complete darkness markedly decreased. CaM-kinase I activity assay supported these findings. Synapsin I was shown to be a possible intrinsic substrate of CaM-kinase I in rat retina. These results elucidated that CaM-kinase I is expressed in the retina and may play an important role in the retinal functions and that the expression of CaM-kinase I is regulated by light stimulation.     

Pharmacokinetics of 9alpha-fluoromedroxyprogesterone acetate in rats: comparison with medroxyprogesterone acetate

Medroxyprogesterone acetate (MPA) is widely used in endocrine therapy for breast cancer and other diseases. Recently, it has been demonstrated that 9alpha-fluoromedroxyprogesterone acetate (FMPA) also has anti-tumour activity in chemical-induced rat mammary tumour and its activity is greater than that of MPA. In the present study, the physico-chemical properties of FMPA and MPA and their pharmacokinetics in female rats were investigated. Partition coefficients (log P) of FMPA and MPA were 3.1 and 3.8, respectively, while the solubilities of FMPA and MPA in phosphate buffer saline were 3.8 and 1.1 microg/mL, respectively. When the two agents were intravenously or orally administered into female rats, there was no significant difference between their plasma concentrations. However, unmetabolized drug excreted into urine accounted for 4.7 and 0.7% of the intravenous dose of FMPA and MPA, respectively. The free fraction of FMPA in rat plasma was approximately four times that of MPA. Assuming the well-stirred model, hepatic intrinsic clearances of FMPA and MPA were estimated to be 64 and 293 L/h per kg, respectively. In addition, the free fraction of FMPA in blood is estimated to be higher than that of MPA, which may explain the higher anti-tumour activity.     

Antitumor activity of HO-221, a derivative of benzoylphenylurea against human cancer xenografts in nude mice

HO-221, a derivative of benzoylphenylurea, is a newly developed anticancer drug which was found to show an excellent antitumor effect against transplantable murine tumors by the novel mechanism of action. This study was designed to evaluate the antitumor effect of HO-221 and to establish the optimum regimen, using seven human gastrointestinal and breast cancers xenografted in nude mice. Better antitumor effect of HO-221 by oral administration was observed when it was suspended in larger volume of the vehicle. Moreover, the effect increased by the multiple intermittent administration compared to the single treatment. Best antitumor effect was observed by oral administration of 75 mg/kg (0.1 ml/10 g mouse body weight) repeated twice weekly for a total of eight times or 300 mg/kg (0.2 ml/10 g mouse body weight) repeated once weekly for a total of four times. The antitumor effects of these two regimens were approximately equal except against H-31, the former regimen being more effective. When the tumor growth inhibition rate (IR) over 58% was rated as "effective", the above two regimens were equally effective against 4 of 7 cancers, H-111, H-154, H-143 and H-31. While HO-221 was not effective to a gastric cancer line, H-81, which was most susceptible to the variety of existing anticancer agents, but effective to another gastric cancer line, H-111, which was relatively resistant to conventional cytocidal agents. From the aspect of chemosensitivity spectrum, this drug revealed a rather different pattern compared to other antimetabolites. Although oral administration volume is limited in small animal model, enhancing its antitumor effect may be possible in clinical application by contriving the method of administration. HO-221 is, thus, considered to be a promising drug for further study.