Presynaptic localization of histamine H3-receptors in rat brain.

The localization of histamine H3-receptors in subcellular fractions from the rat brain was examined in a [3H] (R) alpha-methylhistamine binding assay and compared with those of histamine H1- and adrenaline alpha 1- and alpha 2-receptors. Major [3H](R) alpha-methylhistamine binding sites with increased specific activities ([3H]ligand binding vs. protein amount) were recovered from the P2 fraction by differential centrifugation. Minor [3H](R)alpha-methylhistamine binding sites with increased specific activities were also detected in the P3 fraction. Further subfractionation of the P2 fraction by discontinuous sucrose density gradient centrifugation showed major recoveries of [3H](R)alpha-methylhistamine binding in myelin (MYE) and synaptic plasma membrane (SPM) fractions. A further increase in specific activity was observed in the MYE fraction, but the SPM fraction showed no significant increase in specific activity. Adrenaline alpha 2-receptors, the pre-synaptic autoreceptors, in a [3H] yohimbine binding assay showed distribution patterns similar to histamine H3-receptors. On the other hand, post-synaptic histamine H1- and adrenaline alpha 1-receptors were closely localized and distributed mainly in the SPM fraction with increased specific activity. Only a negligible amount was recovered in the MYE fraction, unlike the histamine H3- and adrenaline alpha 2-receptors.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial specific granules in the umbilical veins of the postnatal rabbit

Summary A remarkable increase in number of endothelial specific granules was observed in the rabbit umbilical veins between 2 and 5 days after birth. Electron microscopy indicated that the granules were segregated in the Golgi complex of the endothelial cells and released into the vascular lumen during the postnatal obliteration stage of this vessel.Incubation of the postnatal vessels in Ringer solution containing a histamine releasing compound induced remarkable morphological alterations of these cytoplasmic components; a reduction of their osmiophilia, swelling with a widened space separating the granular matrix from the limiting membrane, fusion to each other and expulsion of their contents into the vascular lumen, as in mast cell degranulation by this drug, were noted.High-performance liquid chromatography of the homogenized vessels demonstrated appreciable concentrations of histamine in the postnatal samples. There was a correlation between the histamine concentration and the quantity of granules in the respective postnatal samples.The present study strongly suggests that the granules are reservoirs of histamine and have an important role in the obliteration of this vessel.This work was supported in part by Grant in Aid for Scientific Research (# 448087) to S. Fujimoto from the Ministry of Education of Japan     

Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein.

The crp gene of E. coli, which codes for cAMP receptor protein (CRP), has been cloned in the plasmid pBR322 on the basis of a genetic complementation. One of the recombinant plasmids, pHA1, was shown to direct the synthesis of CRP in a cell-free system. The location of the crp gene was determined by constructing subclones carrying various portions of pHA1. The nucleotide sequence of the crp gene has been determined. The coding region consists of 627 base pairs (bp), which specify a protein of 209 amino acids. The predicted amino acid sequence from the DNA sequence is consistent with the amino acid sequence partially known and the amino acid composition of CRP. After the coding region, there is a G-C rich inverted repeat sequence followed by a run of Ts, which could be a terminator of the crp gene. A possible promoter sequence was found about 180 bp upstream from the initiation codon and was shown to act as a promoter in vitro and in vivo. There are two dyad symmetry regions in a 167 bp leader sequence.     

A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (M_r 17 000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.     

Sesquiterpenoids of Riccardia and Pallavicinia species

Riccardia species (Metzgeriales) contain various types of sesquiterpenes. R. jackii produces ent-selinane-, ent-aromadendrane-and ent-bicyclogermacrane-type sesquiterpenes together with (R)-cuparene and α-barbatene. Aneura pinguis (= Riccardia pinguis) is chemically quite different from R. multifida and R. jackii. The former produces a large amount of pinguisone. R. multifida contains 6-(3-methyl-2-butenyl)-indole and (+)-β-elemene as the major components. Pallavicinia longispina (Dilaenaceae; Metzgeriales) produces mainly spathulenol. The chiral properties of the sesquiterpenes isolated from R. jackii are quite similar to those of red algae, Laurencia species.     

Bibenzyls from Radula tokiensis and R. Japonica

A new bibenzyl having a dihydrooxepin ring was isolated from the acetone extract of the liverwort Radula tokiensis, together with the previously known 5 bibenzyls and 3 sesquiterpenes, trans-β-farnesene, cuparene and (Jcuparenol. Two known bibenzyls were isolated from R. japonica. The bibenzyl derivatives are significant chemosystematic markers of the Radulaceae.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.