Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Caenorhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacin-like metalloprotease with an epidermal growth factor (EGF)-like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoter-driven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wild-type rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.     

Novel biodegradable polyphosphate cross-linker for making biocompatible hydrogel

To obtain a novel biodegradable cross-linker, polymerizable polyphosphate (PIOP) was synthesized by ring-opening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl methacrylate) (OPEMA). The number averaged molecular weight of the PIOP was 1.2 x 10(4), and the number of OPEMA units in one PIOP molecule was 2.2. Nonenzymatic degradation of the PIOP was evaluated in various pH aqueous media. The degree of hydrolysis was dependent on the pH; that is, it increased with an increase in the pH of the medium. At pH 11.0, the PIOP completely degraded in only 6 days. The poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] cross-linked with the PIOP was prepared by radical polymerization. This polymer could form hydrogel, and the free water fraction in the hydrogel was high. The enzymatic activity of trypsin in contact with the hydrogel was similar to that in buffer solution. There is no adverse effect caused by the hydrogel to reduce the function of the trypsin. The cytotoxicity of poly(MPC) and degraded PIOP was evaluated using v79 cells, and it was not observed in either case. In conclusion, PIOP is a hydrolyzable polymer, which can be used as a cross-linker, and novel hydrogels having biodegradability and biocompatibility were prepared from poly(MPC) cross-linked with the PIOP.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.     

Influence of Temocapril on Cultured Ventral Spinal Cord Neurons

Temocapril, a angiotensin-converting enzyme (ACE) inhibitor, was tested for neurotrophic activity in primary explant cultures of ventral spinal cord of fetal rats (VSCC). Temocapril had a remarkable effect on neurite outgrowth with a 4.2- to 5.1-fold increased over that of control VSCC at their effective concentrations. In temocapril-treated VSCC, choline acetyltransferase (ChAT) activity was also increased 2.4–3.2 times over that of control at 10^–9 and 10^–8 M, respectively. Our data suggest that temocapril is a candidate for neurotrophic factors on spinal motor neurons in vitro. A possible therapeutic role for temocapril in damaged motor neurons, such as in motor neuropathy and amyotrophic lateral sclerosis, remains to be defined.     

Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.