Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.     

Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different

We have purified putative L-type Ca2+ channels from chick heart by virtue of their associated high affinity receptors for the Ca2+ channel effectors, dihydropyridines (DHPs), and phenylalkylamines (PAAs). A peptide of 185,000-190,000 daltons was found to comigrate with the peak of DHP binding activity during purification through two successive cycles of lectin affinity chromatography and sucrose density gradient centrifugation. A previously described peptide of 140,000 daltons, whose Mr was increased to approximately 180,000 under nonreducing conditions, also copurified with the 185-kDa peptide and dihydropyridine binding activity. When cardiac membranes were photolabeled with either the dihydropyridine [3H]azidopine or the PAA [3H]azidopamil prior to purification, a single, specifically labeled component of 185,000-190,000 daltons was present in the purified fractions. The properties of this 185-kDa cardiac DHP/PAA receptor were compared to the smaller 165-kDa DHP/PAA receptor previously purified from skeletal muscle. Antibodies raised against the 165-kDa skeletal muscle DHP/PAA receptor reacted with both rabbit and chick skeletal muscle receptors, but only poorly recognized, if at all, the cardiac 185-190 kDa component. The 185-kDa peptide present in the purified fractions obtained from cardiac muscle did not undergo substantial phosphorylation by cAMP-dependent protein kinase, while the purified 165-kDa peptide from rabbit and chick skeletal muscle was a good substrate for this kinase. The results show that the DHP and PAA receptors in cardiac muscle are contained in a 185-190-kDa peptide that is significantly larger than, and structurally and immunologically different from, it skeletal muscle counterpart.     

cDNA and deduced primary structure of rat protein B23, a nucleolar protein containing highly conserved sequences

Protein B23 (Mr/pI = 38,000/5.1) is a major RNA-associated nucleolar phosphoprotein which contains highly acidic segments and has a high affinity for silver ions. Using synthetic oligonucleotides as probes cloned cDNAs encoding protein B23 were isolated and characterized. One of the cDNAs, obtained from a rat brain library, contained an insert of 1232 base pairs of DNA encoding a polypeptide of 292 amino acid residues. Segments of the protein sequence were confirmed by partial sequencing of CNBr fragments from rat hepatoma protein B23. The protein contains a methionine-rich amino-terminal sequence and two highly acidic segments in the center of the sequence. The first acidic segment, in which 11 of the 13 residues are acidic, begins at residue 120 and contains a major phosphorylation site. In the second segment (residues 159-187) there are four copies of the sequence Asp-Asp-Glu, and all but two of the 29 residues have acidic side chains. When the sequence of the rat protein was compared with available sequences from other species a high degree of conservation was found; the 77-residue carboxyl-terminal sequence is identical with that of human protein B23 (Chan, P. K., Chan, W.-Y., Yung, B. Y. M., Cook, R. G., Aldrich, M. B., Ku, D., Goldknopf, I. L., and Busch, H. (1986) J. Biol. Chem. 261, 14335-24341), and about 63% of the residues are identical when the rat B23 sequence is compared with protein N038 from Xenopus laevis (Schmidt-Zachmann, M. S., Hügle-D?rr, B., and Franke, W. (1987) EMBO J. 6, 1881-1890). Except for the presence of highly acidic regions no significant similarities were found with protein C23 (nucleolin), the other major nucleolar protein.     

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

Electron Microscopic Studies of Spruce Needles in Connection with the Occurrence of Novel Forest Decline

Needles of four spruce trees showing different degrees of novel kinds of forest decline were investigated by electron microscopy. Green needles appearing at least superficially still intact were selected for the present investigation. Most of the mesophyll appeared to be undamaged. However, groups of atypical mesophyll cells were found close to the endodermis or the hypodermis. The chloroplasts of the apparently damaged cells were particularly affected. Changes in the matrix of the chloroplasts, i.e,. increased affinity to osmium, occurrence of extensive nests of plastoglobuli, as well as damage to the membranes, i.e. lesions in the envelope and abnormal thylakoid membranes, were observed. Signs of decomposition of other cellular structures including mitochondria were also detectable. There appeared to be a close correlation between the degree of damage at the whole tree level and the degree of damage occurring at the cellular level. It is concluded that particularly the lipids and the proteinsof, the membranes are affected by anthropogenic air pollutants and natural stressors. The altered membrane structure may for instance cause abnormal osmotic conditions for the cellular compartments and may impair transport processes and thus lead to lossof function not only of the cells but also of the whole needle.     

植物新药——甘木通的生药鉴别

木文对甘水通的生药结构、药用情况、鉴别点、地理分布及繁殖等进行了报道;同时指出造成混乱的原因及找出混乱种的学名。甘本通为分布于广东和广西两省区的特有种。     

A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.