Sesquiterpenoids from Ferula kuhistanica

Methanol extracts of the air-dried roots and stems of Ferula kuhistanica afforded seven daucane-type sesquiterpenes, called kuhistanicaol A-G, together with 13 known daucane esters. Their structures were established on the basis of spectroscopic evidence and the results of chemical reactions.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Local and global cerebral blood flow and glucose utilization in the α-galactosidase A knockout mouse model of Fabry disease

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.     

Assignment of the Gene for Porcine Insulin-Like Growth Factor Binding Protein 1 to Chromosome 18 and Detection of Polymorphisms in Intron 2 by PCR–RFLP

We have obtained a partial cDNA and three BAC clones for the porcine insulin-like growth factor binding protein 1 gene (IGFBP-1). Results of fluorescence in situ and radiation hybrid (RH) mapping assigned this gene to porcine chromosome (SSC) 18q24-qter. We found two types of polymerase chain reaction–restriction-fragment-length polymorphisms (PCR–RFLP) in intron 2 by using FokI and AluI.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels

Arachidonic acid (AA) is generated via Rac-mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in a time- and dose-dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA-induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA-induced SAPK/JNK activation was significantly inhibited in Rat2-Rac1N17 dominant-negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.     

Activation of big MAP kinase 1 (BMK1/ERK5) inhibits cardiac injury after myocardial ischemia and reperfusion

Big MAP kinase 1 (BMK1/ERK5) plays a critical role in pre-natal development of the cardiovascular system and post-natal eccentric hypertrophy of the heart. Of the two isoforms upstream of MAPK-kinase 5 (MEK5) known to exist, only the longer MEK5alpha isoform potently activates BMK1. We generated cardiac-specific constitutively active form of the MEK5alpha (CA-MEK5alpha transgenic (Tg) mice), and observed a 3 to 4-fold increase in endogenous BMK1 activation and hyperphosphorylation of connexin 43 in the ventricles of the Tg compared to wild-type mice. The CA-MEK5alpha-Tg-mice demonstrated a profoundly accelerated recovery of left ventricular developed pressure after ischemia/reperfusion. We propose a novel role for BMK1 in protecting the heart from ischemia/reperfusion-induced cardiac injury.     

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG. Received: 4 November 1997 / Accepted: 5 January 1998     

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.