Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Formation of vitamin D-binding protein-actin and binary and ternary plasma gelsolin-actin complexes in human serum

After the addition of actin to serum, the binding of actin to serum actin-binding proteins was analyzed by the method of immunoblotting using monospecific antibodies against vitamin D-binding protein (DBP) (group-specific component, Gc), human skeletal actin and human plasma gelsolin. When increasing amounts of globular actin were added to serum, actin bound to DBP preferentially. After exhausting DBP, actin began to bind to plasma gelsolin. When equally increasing amounts of filamentous actin were added to serum, actin was bound to both plasma gelsolin and DBP, and then uncomplexed DBP removed one actin molecule from gelsolin-actin 1:2 complex, resulting in a gelsolin-actin 1:1 complex. These results support the theory that the actin-depolymerizing activity of serum is due to the concerted role of plasma gelsolin and DBP.     

Detection of point mutation in the tyrosinase gene of a Japanese albino patient by a direct sequencing of amplified DNA

Summary Enzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of an albino patient. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient.     

Possible physiological role of guanosine triphosphate and inositol 1,4,5-trisphosphate in Ca2+ release in macrophages stimulated with chemotactic peptide.

The release of Ca2+ induced by inositol 1,4,5-trisphosphate (InsP3) in the presence of GTP was examined by using saponin-permeabilized macrophages. The origin and the amount of mobilized Ca2+ in intact macrophages stimulated with chemotactic peptide were also examined to assess the physiological significance of GTP and InsP3 on Ca2+-releasing activities. The total amount of Ca2+ released by 20 microM-A23187 from the unstimulated intact macrophages was 1.4 nmol/4 x 10(6) cells, and the mitochondrial uncoupler did not cause an efflux of Ca2+ from the cells. The Ca2+ accumulation by the non-mitochondrial pool(s) was inhibited by the presence of GTP, and the total amount of releasable Ca2+ (1.4 nmol/4 x 10(6) cells) was comparable with that accumulated by the non-mitochondrial pool(s) in the presence of GTP at a free Ca2+ concentration of 0.14 microM. The mobilized and subsequently effluxed Ca2+ in cells stimulated with chemotactic peptide was estimated to be 0.3 nmol/4 x 10(6) cells. Much the same amounts were released by about the half-maximal dose of InsP3 from the non-mitochondrial pool(s) of saponin-treated macrophages that had accumulated Ca2+ at a free concentration of 0.14 microM in the presence of GTP. These results suggest that the Ca2+-releasing activity induced by GTP may play a role in the long-term regulation of Ca2+ content in the non-mitochondrial pool(s) of macrophages, and that released by InsP3 can explain, quantitatively, the chemotactic-peptide-induced mobilization of Ca2+.     

Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

Effect of streptozotocin-induced diabetes on the activity of 7 alpha-hydroxy-4-cholesten-3-one-specific 12 alpha-hydroxylase in rats

The activity of 12 alpha-hydroxylase in hepatic microsomes from normal, streptozotocin-induced diabetic, and insulin-treated diabetic rats was studied with 7 alpha-hydroxy-4-cholesten-3-one as a substrate. In the diabetic rats, the 12 alpha-hydroxylase activity was about 50% lower than in the normal rats. Treatment of the diabetics with insulin cancelled the reduction of the activity. These results show that an insulin-deficient state causes a paradoxical decrease in the activity of the key enzyme for cholic acid formation.     

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.