Mutual interactions between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between the bilaterally paired optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording locomotor activity, under constant light or constant red light, after the optic nerve was unilaterally severed.

Cloning and expression of cDNAs from enterically-transmitted non-A, non-B hepatitis virus.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of 20(R),22(R)-dihydroxycholesterol and reduced adrenodoxin

Electron paramagnetic resonance (EPR) spectra of ferrous-nitric oxide (14NO and 15NO) cytochrome P-450scc complexed with 20(R),22(R)-dihydroxycholesterol were measured at 77 K with X-band (9.35 GHz) microwave frequency. The EPR spectra clearly showed the spin system to have rhombic symmetry (gx = 2.068, gz = 2.001, gy = 1.961, and Az = 1.89 mT for 14NO) and were distinct from those of 20(S)-hydroxycholesterol complexes. The unique nature of the 20(S)-hydroxycholesterol complexes indicates that 20(S)-hydroxycholesterol is not a proper intermediate in the cholesterol side-chain cleavage reaction. In addition, among various steroid complexes of ferrous-NO species having rhombic symmetry, the EPR spectra of 20(R),22(R)-dihydroxycholesterol complexes were significantly different from those of 22(R)-hydroxycholesterol complexes, suggesting that upon 20S-hydroxylation of 22(R)-hydroxycholesterol the conformation of the active site changes so as to facilitate subsequent cleavage of the C20-C22 bond of the cholesterol side chain. Addition of reduced adrenodoxin to the ferrous-NO cytochrome P-450scc complex in the presence of cholesterol caused a complete shift of the gx = 2.070 signal to gx = 2.075, indicating a reorientation of cholesterol in the substrate-binding site of the enzyme upon adrenodoxin binding. Without reduced adrenodoxin, the process of reorientation of cholesterol in the substrate-binding site was very slow, requiring more than 50 h of incubation at 0 degrees C. The present observations suggest that adrenodoxin may have another positive role in the cholesterol side-chain cleavage reaction, in addition to transferring an electron to the heme of cytochrome P-450scc.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.     

Heterogeneity of adrenocortical ferredoxin

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.     

Separation of DNA binding domain from hormone and core histone binding domains by trypsin digestion of rat liver nuclear thyroid hormone receptor

Rat liver nuclear thyroid hormone receptor was subjected to limited trypsin digestion, and the tryptic fragment of the 3,5,3'-triiodo-L-thyronine (T3)-receptor complex was characterized. Rat liver nuclear thyroid hormone receptor is an asymmetrical protein with Stokes radius of 34 A, sedimentation coefficient of 3.4 S, and molecular weight of 49,000. A globular T3-receptor complex with Stokes radius of 22 A, sedimentation coefficient of 2.8 S, and molecular weight of 26,000 was obtained by tryptic digestion. This fragment had no DNA binding activity, whereas undigested receptor showed significant DNA binding activity. Addition of undigested receptor to the tryptic fragment did not restore DNA binding activity of digested receptor, nor did mixing inhibit DNA binding activity of undigested receptor complex. Undigested receptor bound to core histones, and this activity was stronger than with other proteins tested (H1 histone, cytochrome c, and ovalbumin). The tryptic fragment of receptor maintained core histone binding activity comparable to that of undigested receptor. The tryptic fragment had affinity for T3 comparable to undigested receptor as assessed by Scatchard analysis and the same rate for dissociation of [125I]T3 from receptor. The tryptic fragment of the T3-receptor complex was more stable than undigested receptor at 43 degrees C. Digestion of receptor unoccupied by T3 caused a significantly larger loss of T3 binding capacity than did digestion of T3-occupied receptor, suggesting a protective effect of T3 on a second trypsin-sensitive site on the receptor, which, when cut, destroys T3 binding activity.     

Resonance Raman detection of a v(Fe-CO) stretching frequency in cytochrome P-450scc from bovine adrenocortical mitochondria

Resonance Raman scattering experiments on CO-complexed cytochrome P-450scc from bovine adrenocortical mitochondria demonstrate the simultaneous enhancement of v(Fe-CO) stretching and bound v(C-O) stretching frequencies at 477 and 1953 cm-1, respectively. These assignments were made on the basis of frequency shifts with the isotope 12C18O. This unusually low v(Fe-CO) stretching frequency in cytochrome P-450scc, compared with other CO-complexed hemoproteins such as CO-hemoglobin and -myoglobin, is presumably due to the thiolate ligation to the heme iron trans to CO and due to the linear and perpendicular configuration of CO binding to the heme.     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.