Structure-activity relationship of benzo[b]thiophene-2-sulfonamide derivatives as novel human chymase inhibitors

We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously discovered by in silico screening. TY-51076 (7) showed high potency (IC(50)=56 nM) and excellent selectivity for chymase compared to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure-activity relationship of this class are described.     

Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5-21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5-5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.     

Calmodulin reverses rundown of L-type Ca(2+) channels in guinea pig ventricular myocytes

Calmodulin (CaM) is implicated in regulation of Ca²⁺ channels as a Ca²⁺ sensor. The effect of CaM on rundown of L-type Ca²⁺ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca²⁺ channel activity disappeared within 1–3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 µM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca²⁺ concentrations ([Ca²⁺]); however, increase to micromolar [Ca²⁺] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca²⁺ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 µM) + ATP induced Ca²⁺ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5'-(,-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1–10 µM) did not block the effect of CaM, indicating that the effect of CaM on the Ca²⁺ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca²⁺ channel activity, showing a cooperative effect of CaM and ATP on the Ca²⁺ channel. These results suggest that CaM is a crucial regulatory factor of Ca²⁺ channel basal activity. cardiac myocyte; calcium channel; patch clamp     

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.     

Small angle X-ray scattering study of the yeast prion Ure2p

The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants Delta 15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP). The native state is globular and predominantly dimeric prior to the onset of unfolding. R(g) values of 32 and 45A were obtained for the native and 5M GdmCl denatured states of Delta 15-42Ure2, respectively; the corresponding values for 90Ure2 were 2-3A lower. SAXS suggests residual structure in the 4M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions. Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions. The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation.     

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.     

Molecular properties of endogenous RFamide-related peptide-3 and its interaction with receptors

Based on database searches of DNA sequences, we previously reported a gene encoding peptides possessing Arg-Phe-NH(2) (RFamide) at their C termini. This gene, RFamide-related peptide (RFRP), was expected to encode several different peptides (i.e., RFRP-1, -2, and -3). In the present study, we purified endogenous RFRP-3 from bovine hypothalamus, and demonstrated that it consisted of 28 amino acid residues. After constructing a sandwich enzyme immunoassay for RFRP-3, we analyzed the tissue distribution of endogenous RFRP-3 in rats and found its concentration to be highest in the hypothalamus. In binding assays, [125I]-labeled RFRP-3 bound to OT7T022 with high affinity, but its binding affinity to HLWAR77 was low. On the other hand, [125I]-labeled neuropeptide FF (NPFF) bound to both OT7T022 and HLWAR77 with high affinity. By serial deletion in the N-terminal portions of RFRP-3 and NPFF, we found that four C-terminal amino acid residues (i.e., PQRFamide), which were common between the two peptides, comprised a core sequence responsible for binding with the receptors, whereas three amino acid residues (i.e., PNL in RFRP-3 and LFQ in NPFF) added to the N terminus of PQRFamide played crucial roles in the agonistic activities of RFRP-3 and NPFF for OT7T022 and HLWAR77, respectively.     

Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae

Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nm, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 microm), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.     

First investigation of ultraoligotrophic alpine Lake Puma Yumco in the pre-Himalayas,China

Lake Puma Yumco is a typical alpine lake (altitude 5030m) located in the pre-Himalayas of Tibet, China, and this study was the first limnological investigation ever conducted on it. Lake Puma Yumco (28°34N, 90°24E) has the following morphometric properties: maximum length 31km, maximum width 14km, mean width 9km, shoreline 90km, surface area 280km², and shoreline development 1.5. Transparency was approximately 10m, even in the thawing season. The extinction coefficient of the lake water was calculated as 0.15m^–1. Annual maximum transparency was estimated from the depth of the Chara zone to be 30m. Dissolved oxygen was 7mg O₂ l^–1 and showed saturated values, and salinity was 360mgl^–1. The chemical type of the lake water was Mg-Ca-HCO₃-SO₄, and it was slightly alkaline in character. Total nitrogenous nutrients (sum of ammonia, nitrite, nitrate, and urea nitrogen), phosphate, and silicate were extremely low at 1, 0.02, and 9µM, respectively. Dissolved organic carbon, nitrogen, and phosphorus concentrations were 160, 11, and 0.08µM and the molar ratio was calculated as 2100:140:1. Chlorophyll a concentration was 0.2mgm^–3. Phytoplankton and zooplankton were dominated by Aphanocapsa sp. and Diaptomidae. Both nitrogen and phosphorus appear to be the limiting parameters for phytoplankton growth. Organic carbon and nitrogen contents in lake sediments were low and the sediments contained a large amount of CaCO₃. The grain size of sediment was that of silt-sand in most cases. The present results indicate that the pre-Himalayan alpine freshwater Lake Puma Yumco is an ultraoligotrophic lake.