Preliminary crystallographic data for plastocyanins from an alga (Enteromorpha prolifera) and from cucumber (Cucumis sativus).

The plastocyanins from a green alga (Enteromorpha prolifera) and cucumber (Cucumis sativus) have been crystallized. Crystal data are as follows: E. prolifera plastocyanin, space group I4, a = b = 53.9 A, c = 59.4 A, Z = 8; C. sativus plastocyanin, space group P4(1) (or P4(3) ), a = b = 66.7 A, c = 46.0 A, Z = 8. Accordingly, the asymmetric units of the crystals contain one and two molecules, respectively.

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera.

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.

Effect of flavones on rat brain and lung matrix metalloproteinase activity measured by film in-situ zymography.

We have evaluated the inhibitory activity of flavone, nobiletin, and heptamethoxyflavone on matrix metalloproteinase (MMP) activity in the rat. MMP in 9000-g supernatant fraction of lung homogenate was activated by p-aminophenyl mercuric acetate (APMA), and gelatinolytic activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. This activity should be related to MMP-2 and/or MMP-9 and was confirmed by gelatin zymography. Fluorescent-conjugated collagen used as a substrate for collagenolytic activity wasinvestigated by SDS-PAGE also. The film in-situ zymography method was applied to rat brain and lung tissue in the same manner. Flavone and nobiletin inhibited the APMA-stimulated gelatinolytic activity and also the collagenolytic activity by more than 75%. The film in-situ zymography method indicated that these compounds might be potent inhibitors of MMP, suggesting the specific inhibition of localized MMP in brain hippocampus and/or lung terminal bronchioles, which may contribute to the prevention of some types of brain disease or cancer invasion and metastasis.

Purification, crystallization, and properties of plastocyanin from a green alga, Enteromorpha prolifera.

Plastocyanin was extracted from a green alga, Enteromorpha prolifera, and purified to an electrophoretically homogeneous state. The ratio of plastocyanin to chlorophyll was 1 : 410. The protein was crystallized; this is the first time that algal plastocyanin has been crystallized. The oxidized form showed absorption maxima at 259, 277, 283.5, 460, 597, and 775 nm, and shoulders at around 253, 265, and 269 nm. The extinction coefficient at 597 nm was 4.7 mM-1.cm-1. The best A277/A597 ratio was 1.3. The maximum at 597 nm shifted to 592.5 nm at 77K. The midpoint redox potential of the plastocyanin was +0.369 volt at pH 7.0 and 26 degrees C. A one-electron change was involved. The molecular weight, estimated by gel filtration, was 12,000, though a minor component with a molecular weight of 22,000 was also observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.1 for the oxidized form. The amino acid composition was: Asp15, Thr6, Ser4, Glu6, Pro5, Gly13, Ala12, Cys1, Val11, Met2, Ile7, Leu4, Tyr3, Phe4, Lys4, His2, Arg1, Trp1, giving a total of 101 residues. Enteromorpha plastocyanin was compared with other plastocyanins from various plants.

The syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational level in a green alga, Pediastrum boryanum.

The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper concentration of the medium. We studied the levels at which the syntheses of the two proteins are regulated. Plastocyanin and cytochrome c-553 were purified from P. boryanum NIES-301 cells, having apparent molecular weights of 14,600 and about 12,000, respectively. Western blotting with antisera raised against these proteins showed accumulation of (apo)plastocyanin and (apo)cytochrome c-553 in the cells grown with (2 microM) and without added CuSO4, respectively, but no accumulation of the precursor proteins in both cultures. The translatable mRNAs for the two proteins were examined by in vitro translation with total RNA and wheat germ extract followed by immunoprecipitation and SDS-PAGE. The 21-kDa polypeptide (preapoplastocyanin) was detected with anti-plastocyanin serum in copper-sufficient cells; the 23-kDa polypeptide (preapocytochrome c-553) with anti-cytochrome c-553 serum in copper-deficient cells. The translatable mRNA for preapoplastocyanin appeared in 1 h and (apo)plastocyanin in 2-3 h after the addition of 2 microM CuSO4 to the copper-deficient culture. The translatable mRNA for preapocytochrome c-553 disappeared within 4-5 h, while (apo)cytochrome c-553 disappeared more slowly. It is concluded that the syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational (i.e., transcriptional or post-transcriptional) level in P. boryanum NIES-301.

Studies on algal cytochromes. I. Purification and properties of cytochrome b-561 from Enteromorpha prolifera.

Cytochrome b-561 (Enteromorpha prolifera) was extracted from a green alga, E. prolifera, by immersion of the dried thalli in phosphate buffer solution. Purification was carried out by ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, Bio-Gel gel filtration, and hydroxyapatite column chromatography. The reduced form of the cytochrome exhibited absorption maxima at 561 (alpha), 530.5 (beta), and 428.5 nm (gamma), and the oxidized form at 530.5, 417 (gamma), and 275 nm. The alpha-band of the reduced form was symmetric without any shoulder. The pyridine hemochrome showed absorption maxima at 556, 524, and 418 nm. The cytochrome does not combine with carbon monoxide or cyanide. The cytochrome showed little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Ferrocyanide and hydroquinone do not completely reduce it. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.23 volt at pH 7.0 and 25 degrees C. The molecular weight of the cytochrome, estimated by Sephadex gel filtration, was 67 x 10(3).

Some properties and amino acid sequence of plastocyanin from a green alga, Ulva arasakii.

Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.

Studies on algal cytochromes. V. Purification and characterization of cytochrome c-552 from a red alga, Polysiphonia urceolata.

Cytochrome c-552 was extracted from a red alga, Polysiphonia urceolata, by immersion of the frozen trichomes in deionized water. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography, hydroxyapatite column chromatography, and Bio-Gel P-10 gel filtration. The ferrocytochrome c-552 has absorption maxima at 551.5(alpha), 522.5(beta), 416.3(gamma), 318(delta), 292, and 270 nm; those of the ferricytochrome are at 525, 408.5(gamma), and 358 nm. The pyridine ferrohemochrome showed absorption maxima at 550(alpha), 520(beta), and 414 nm(gamma). The alpha-band of the ferrocytochrome is symmetric without any shoulder at room temperature, and does not split even at liquid nitrogen temperature. The ferricytochrome showed a weak absorption shoulder at 695 nm, suggesting a methionine sulfur to be the sixth ligand of heme c iron. The cytochrome is oxidized by ferricyanide and reduced by ferrocyanide, cysteine, ascorbate, and hydrosulfite. Autoxidation was not observed. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferricyanide system to be 0.333 volt at pH 7.0 and 25 degrees C. The isoelectric points of the ferro- and ferricytochromes were determined to be at pH 3.85 and 4.02, respectively, by the isoelectric focusing method. The molecular weight was estimated to be about 12,000 from the results of gel filtration and SDS polyacrylamide gel electrophoresis.

Analysis of swertiamarin in Swertia herb and preparations containing this crude drug by capillary electrophoresis.

Swertia herb (florescent whole plantof Swertia japonica, Gentianaceae) has long been utilized as a folk medicine in Japan. It is often blended in general gastroenteric drugs as a bitter stomachic. Swertiamarin, a bitter secoiridoid glycoside, is the representative constituent of this crude drug and Swertia herb is normally evaluated by the swertiamarin content. To date, papers have described the discrimination of Swertia herbs from other bitter crude drugs, estimation of swertiamarin and seasonal variation in swertiamarin content using thin-layer chromatography, while other papers have reported quantitative determination of swertiamarin using high-performance liquid chromatography (HPLC). In our previous papers, we reported analyses of the constituents of some crude drugs using capillary electrophoresis (CE). To aid in the evaluation of crude drugs, we succeeded in our attempt to separate and determine the quantity of swertiamarin in Swertia herb. Subsequently, we applied the same analytical condition to estimate the swertiamarin contents in Japanese pharmacopoeia stomachic preparations, in OTC gastroenteric drugs and in OTC hair tonics containing Swertia herb.

Increase in monoamine levels caused by emotional stress in mice brain regions is attenuated by Saiko-ka-ryukotsu-borei-to.

The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced increase of monoamines in brain regions was investigated in three mouse emotional stress models. Dopamine (DA) and 3,4-dihydroxy-phenyl acetic acid (DOPAC) contents were elevated significantly by electric shock stress, psychological stress and conditioned fear stress in thalamus, hypothalamus and amygdala. The DA and DOPAC levels were decreased by preadministration of SRBT (600 mg/kg, p.o.) in the last two models, but were not altered in electric shock stress. Therefore, this compound seems to be effective in stress involving emotional factors. These results indicate that SRBT affects the brain monoamine neurons leading to psychological change in mice.

[The anti-Candida activity of shikon].

Antifungal activity of Shikon, roots of Lithospermum erythrorhizon and Arnebia euchroma was investigated in vitro. The extracts containing the pigments of Ko-shikon or Nan-shikon showed the antifungal activities against Candida albicans. Acetylshikonin, one of these pigments, inhibited the fungal growth at MIC 15.6 micrograms/ml (RPMI24 h) or 3.9 micrograms/ml (YNB24 h).

[Effect of Oren-gedoku-to on changes in hexobarbital-induced sleeping time in chlorpromazine-treated mice].

The effects of Oren-gedoku-to on the hypnotic duration induced by hexobarbital, and on the change of this duration in chlorpromazine-treated mice were investigated. Single dose of Oren-gedoku-to (1.0 or 2.0 g/kg, p.o.) or chlorpromazine (10 mg/kg, p.o.) prolonged the hexobarbital hypnosis time. And the simultaneous dose of chlorpromazine (6.0 mg/kg, p.o.) and Orengedoku-to (1.0 g/kg, p.o.) indicated the equal effect for the administration of chlorpromazine (10.0 mg/kg, p.o.) alone. Although the mechanism of the action remains to be unknown, administration of Oren-gedoku-to in combination with chlorpromazine seems to be useful from the standpoint of alleviation of the side-effects caused by less dose.

[The mechanism of alteration of monoamine metabolism in brain regions in marble burying behavior-isolated housing mice and effect of oren-gedoku-to on this alteration].

The mechanism of the alteration in marble burying behavior-isolated housing (MBB-IH) mice was investigated. The determination of hypothalamus monoamine and serum corticosterone contents indicated that MBB-IH mice readily responded to the stress stimuli in conditioned fear stress. Six drugs, such as buspirone (10 mg/kg, p.o.), zimelidine (10 mg/kg, p.o.), clomipramin (10 mg/kg, p.o.), yohimbine (5 mg/kg, p.o.), ethyl beta-carboline-3-carboxylate (beta-CCE, 5 mg/kg, p.o.) and flumazenil (15 mg/kg, p.o.) were singly and/or three times administered to MBB-IH mice. Their inhibitory activity on the MBB-IH mice was considered by the use of activity profiles consisting of spontaneous locomotor activity, marble burying behavior and hypothalamus monoamine content. Using these profiles, we calculated the activities as vector in three-dimensional space, and compared the distance from the control point (DCP). DCPDOPAC and DCP5-HIAA were shortened by single administration of beta-CCE and flumazenil. Oren-gedoku-to (30 and 300 mg/kg, p.o.) shortened the DCPDOPAC and DCP5-HIAA similarly to beta-CCE. The blended crude drugs in Oren-gedoku-to, Coptis rhizome (636.0 mg/kg, p.o.), Scutellaria root (644.4 mg/kg, p.o.) and Gardenia fruit (894.8 mg/kg, p.o.) shortened the DCPDOPAC. Coptis rhizome and Scutellaria root also shortened the DCP5-HIAA. These results suggest that GABA neuron function intensely affects the alteration of MBB-IH and Oren-gedoku-to has the intrinsic benzodiazepine-like activity.

Augmentation of the pharmacological action of corydalis tuber by saussurea root in isolated mouse ileum.

To understand the meaning of blending crude drugs in Chinese medicinal prescriptions, the influence of Saussurea root on the pharmacological action of Corydalis tuber was examined. Saussurea root increased the depression of acetylcholine-induced contraction caused by the hot water extract solution of Corydalis tuber in mouse ileum at low dosage, which showed no direct influence on acetylcholine. Dehydrocostuslactone in Saussurea root was characterized as the component having increasing activity and the relationship between the concentration of acetylcholine and the variation in the contraction depressed by Corydalis tuber alone or a mixture of the Corydalis tuber and dehydrocostuslactone was investigated for clarification of the mode of action.

Accumulation of plastocyanin mRNA lacking 5' region in the green alga Pediastrum boryanum grown under copper-deficient conditions.

In the green alga Pediastrum boryanum NIES-301, plastocyanin accumulates under copper-sufficient conditions and cytochrome c6 accumulates under copper-deficient conditions. We cloned the cDNA which encodes pre-apoplastocyanin from P. boryanum cultured under the copper-sufficient condition. The deduced amino acid sequence of the pre-apoplastocyanin protein consists of 151 amino acid residues including a putative bipartite presequence of 53 amino acid residues. Southern blot analysis of P. boryanum genomic DNA indicated that pre-apoplastocyanin is encoded by a single nuclear gene. Northern blot analysis showed that copper-deficient cells accumulated a shorter form of the mRNA of pre-apoplastocyanin, which did not generate pre-apoplastocyanin in the wheat-germ translation system. The difference in size was ascribed to the absence of the 5' region in the mRNA of pre-apoplastocyanin obtained from the copper-deficient cells, which accounts for the absence of plastocyanin under these conditions. This phenomenon represents a novel regulatory mechanism, although details of the mechanism are not yet known.

[The medicine chest handed down in the Takaya family, doctors of Sendai-han].

There is a medicine chest with design of Chrysanthemums in Makie made for Edo-era and handed down in the Takaya family, doctors of Sendai-han. Many kinds of crude drugs, named by one Chinese character, are kept in this chest. They were classified into three types, namely, [Japanese characters]. Identification of these crude drugs was carried out and the meaning of the classification was brought up as a question.

Effect of saiko-ka-ryukotsu-borei-to on the stress-induced increase of serum corticosterone in mice.

The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced enhancement of serum corticosterone in various stress models was investigated in mice. The serum corticosterone was elevated significantly by immobilized stress, forced-swim stress, electric-shock stress, psychological stress and conditioned-fear stress, respectively. The concentration in the last two models was decreased in a dose-dependent manner by pre-administration of SRBT (10, 60, 100, 600 and 1000 mg/kg, p.o.). Therefore, this action seems to be effective in stress involving emotional factors but ineffective in physical stress models. These results indicate that the anti-stress effect of SRBT is dependent strongly on the degree of psychological change compared with physical change in mice.

[Evaluation of marble burying behavior: induced alteration of monoamine metabolism in mouse brain].

Evaluation of marble burying behavior (MBB) housing-induced alteration of monoamine metabolism in mouse brain was performed by measuring metabolite levels with HPLC-ECD. Isolated housing of mice, each in a cage (22 x 32 x 14 cm; sawdust, 1-mm diameter; 5 cm in thickness) with 15 evenly spaced glass marbles on the floor (2.5-cm diameter; control, without marbles) for 24-168 hr, 5-HIAA contents were decreased in three regions: the midbrain, thalamus and hypothalamus. 5-HT turnover was not inhibited except for in the hypothalamus due to the decreases of 5-HT in the other two regions. On the other hand, DOPAC content and DA turnover were decreased in four regions: striatum, midbrain, thalamus, hypothalamus. The decrease in hypothalamic monoamine neurons was observed notably after 72 hr of MBB housing. No alterations were observed in feeding, water-drinking, spontaneous locomotor activity, number of buried marbles, serum corticosterone and serum glucose concentrations during MBB keeping compared with the control mice. These results suggested that the isolated mouse housed in a cage with evenly spaced glass marbles for a long period may be a model animal for alteration of monoamine metabolism in brain regions without physical infringement.