Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots,and its immunocytochemical localization in dermal tissues

A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M _r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M _r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same M_r and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GP57 M _r-57000 glycoprotein - GP65 M _r-65000 glycoprotein - MAb monoclonal antibody - M _r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TFMS trifluoromethanesulfonic acid     

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores

Abstract Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and were tentatively identified as Chromatium purpuratum . Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of intracellular localization of antibiotic activity in a marine bacterium.     

Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Intracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Two soluble glycoproteins containing hydroxyproline were extractedfrom cultured tobacco cells (cell line XD-6S) and purified byion-exchange and gel-filtration chromatography. On DEAR-cellulosecolumn chromatography in the final step of the purification,one was eluted at 90 mM NaCl and the other at 120 mM as singlepeak. Both purified glycoproteins were also sedimented as singlepeak with an ultracentrifugation. The S_20,w values were 6.1for the former and 7.0 for the latter. These glycoproteins were composed of 94% polysaccharide and6% protein in the former, and 87% polysaccharide and 13% proteinin the latter. The sugar moiety consisted of galactose, arabinose,rhamnose, and uronic acid in both. Hydroxyproline accountedfor 12% in the former and 20% in the latter amino acid composition.A high content of alanine in both (14 and 15%) was one of thedistinctive characteristics of these soluble glycoproteins. These intracellular soluble hydroxyproline-containing glycoproteinswere not labelled within 30 min of incubation with ₃H-proline,although the radioactivity was rapidly incorporated (within15 min) into the intracellular macromolecules. (Received February 21, 1978; )     

C-reactive protein-induced upregulation of extracellular matrix metalloproteinase inducer in macrophages: inhibitory effect of fluvastatin

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMP)-9 were reported to be expressed at the macrophage-rich area in human coronary atherosclerotic plaque. We examined whether C-reactive protein (CRP) activates macrophages to express EMMPRIN and MMP-9 in vitro and whether statins inhibit it. METHODS AND RESULTS: Rat peritoneal macrophages were collected by peritoneal lavage, and were incubated in the presence or absence of CRP. CRP at 5 microg/ml increased the gene expression of EMMPRIN relative to GAPDH, measured by RT-PCR, by 1.67+/-0.07 fold at 24 h and by 1.85+/-0.49 fold at 48 h (both p<0.05). The gene expression of MMP-9 in the presence of CRP at 5 microg/ml was followed by 1.36+/-0.11 fold increase at 24 h and by 3.95+/-0.81 fold at 48 h (both p<0.05). CRP at 5 microg/ml for 48 h increased by 6 fold MMP-9 activity, measured by zymography, without affecting tissue inhibitor of metalloproteinases-1. Boiled CRP at 5 mug/ml for 48 h unaffected MMP-9 activity. Fluvastatin blocked the CRP-induced increases in EMMPRIN and MMP-9 expression and activity. Diphenylene iodonium, an inhibitor of NADPH oxidase, had a similar effect on MMP-9 activity. Fluvastatin suppressed the CRP-induced increases in 8-epi-prostaglandin F(2alpha) levels in the condition media. CONCLUSIONS: CRP is an activator for macrophages to enhance EMMPRIN and MMP-9 expression. Fluvastatin inhibits them presumably through its antioxidant effect.     

Binding site on human von Willebrand factor of bitiscetin,a snake venom-derived platelet aggregation inducer

Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans, binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to botrocetin. In contrast with botrocetin which binds to the A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be a bitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor botrocetin had an inhibitory effect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWF monoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to alpha4 helix of the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilized bitiscetin was competitively inhibited by a high concentration of botrocetin. A panel of recombinant VWF, in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1 domain, showed that the bitiscetin-binding activity was reduced in mutations at Arg632, Lys660, Glu666, and Lys673 of the A1 domain. Those substituted at Arg629, Arg636, and Lys667, which decreased the botrocetin binding, showed no effect on the bitiscetin binding. These results indicate that bitiscetin binds to a distinct site in the A1 domain of VWF spanning over alpha4a, alpha5 helices and the loop between alpha5 and beta6 but close to the botrocetin- and NMC-4-binding sites. Monoclonal antibodies recognizing the alpha-subunit of bitiscetin specifically inhibited bitiscetin-induced platelet agglutination without affecting the binding between VWF and bitiscetin, suggesting that the alpha-subunit of bitiscetin is located on VWF closer to the GPIb-binding site than the beta-subunit is. Bitiscetin and botrocetin might modulate VWF by binding to the homologous region of the A1 domain to induce a conformational change leading to an increased accessibility to platelet GPIb.