Gibberellin Production in Cultured Cells of Nicotiana tabacum

Endogenous gibberellins (GAs) in several kinds of crown gallcells and cultured cells derived from normal tissue of Nicotianatabacum were systematically analyzed by gas chromatography-selectedion current monitoring (GC-SICM) after chromatographic purifications,and GA₁, GA₉, GA₁₉ and GA₂₀ were identified. Agrobacterium tumefaciens,a pathogen of crown gall, was confirmed not to produce GAs inits culture. We also investigated endogenous GAs of mother plant,tobacco, and found the same kinds of GAs as in cultured cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received May 19, 1982; Accepted July 22, 1983)     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Reconstitution mechanism of nucleosome core particles mediated by poly(l-glutamic acid)

Poly(l-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5000–5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(l-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(l-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(l-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo ‘nucleosome assembly factors’ is also discussed.     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Separation of the Polypeptides of Chlamydia and Its Cell Walls by Polyacrylamide Gel Electrophoresis

The polypeptide composition of Chlamydia was examined by acrylamide gel electrophoresis. When the polypeptide patterns of purified infectious elementary bodies (EB) of C. psittaci meningopneumonitis strain, 6BC strain, and C. trachomatis T'ang strain were compared, no significant differences were observed. The polypeptide patterns of whole EB and reticulate bodies (RB) appeared to overlap, but differences were found. In EB cell walls, nine main and several minor bands of polypeptides were observed in gels containing sodium lauryl sulfate, and the eighth main band from the top of the gel stained positive with periodic acid-Schiff reagent. On the other hand, the polypeptides in bands 3, 6, and 8 in EB cell walls were missing or minor in RB cell walls, and the ninth band was clearly stained by PAS. Band 8 was also stained slightly. Purified subunits, which occur as a lattice structure on the inside layer of EB cell walls but are largely missing in RB cell walls, contained bands 4, 6, and 8, and band 8 was PAS positive. These results indicate that significant polypeptide synthesis or reorganization in the cell walls occurs during the growth cycle.     

Dielectric properties of polyelectrolytes. V. Dielectric dispersion of heavy meromyosin

Dielectric constant and dielectric loss of heavy meromyosin (HMM) were measured with varying pH. HMM showed a broader dispersion pattern than that with a single relaxation time especially on the high-frequencey side. The dielectric increment increased sharply with pH, above p^{H 6}, whereas the mean relaxation time and whole dispersion pattern were unchanged in the same region. The values of the increment and the mean relaxation time were much larger than those of usual globular proteins. The dispersion profile, pH dependence, and values of the increment are well explained by Oosawa's counterion fluctuation theory. Other mechanisms are more or less inadequate to our results. In the low pH region below the isoelectric precipitation region, both the increment and the mean relaxation time decreased; this is probably due to partial denaturation and suppression of the dissociation of carboxyl groups. An experiment on a urea-denatured sample supports this assumption. The biological significance of the pH dependence is discussed.     

Distribution and serological specificity of sialidase produced by various groups of streptococci

The occurrence of a streptococcal sialidase (designated St-sialidase) in culture fluids of various streptococci was investigated. St-sialidase was found to occur in strains belonging to groups A, B, C, E, G, H, and L, and the unclassified strains, Streptococcus sanguis and Streptococcus uberis. St-sialidase of group A was confined predominantly to types 4 and 22. St-sialidases, extracted from the culture fluids of some selected strains, were antigenic, eliciting the formation of antibody which effectively neutralized the enzymatic activity of the enzyme. Antisera to the St-sialidases of groups A, B, C, E, G, and L, and Streptococcus sanguis were produced in rabbits. The St-sialidases of groups A, B, and E streptococci were serologically distinct and group-specific. The St-sialidases from groups C, G, and L were serologically homologous, but distinct from St-sialidases of the other groups. Antiserum to the enzyme of strain 10557 (S. sanguis) cross-reacted with the St-sialidase of strain 9927 (S. uberis).     

Purification and Chemical Composition of Reticulate Bodies of the Meningopneumonitis Organisms

Reticulate bodies of the meningopneumonitis (MP) microorganism were purified from L cells 18 hr after infection by the combination of differential centrifugation in 30% sucrose solution and potassium tartrate density gradient centrifugation. It was ascertained by electron microscopy that purified preparations of reticulate bodies obtained were almost entirely free of host-cell components and of infectious elementary bodies of MP microorganisms. Purified reticulate bodies were easily disrupted by mechanical agitation, and it was observed in shadowed preparation that ribosome-like particles 15 mmu in diameter were scattered from broken reticulate bodies. In shadowed preparations, reticulate bodies were found to range in size from 1.0 to 1.6 mu in diameter, but in cross-section the range was 0.5 to 1.0 mu. In these preparations, the purified reticulate bodies were irregular in shape, round or oval, and were composed of rather homogenous, amorphous, or reticulate material with moderate density. Some particles exhibited a less-dense internal structure, in which a coarse fibrous reticulum was seen. Chemical fractionation of (32)P-labeled purified reticulate bodies showed that they contained three times more ribonucleic acid (RNA) than deoxyribonucleic acid, with the RNA being composed primarily of 21S, 16S, and 4S RNA. No infectivity of purified reticulate bodies could be demonstrated.