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)     

Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2

Background

Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated.

Methods

We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out.

Results

We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules.

Conclusions

Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2.

General Significance

This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.     

Catalytic flexibility of glycosylases. The hydration of maltal by beta-amylase to form 2-deoxymaltose

Crystalline, alpha-glucosidase-free sweet potato beta-amylase was found to catalyze hydration of the enolic bond of maltal (alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-glucal) to form 2-deoxymaltose (alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-glucose). The reaction at pH 5.0 showed Vmax 0.082 mumol/min/mg and km 94.5 mM. An exceptionally large solvent deuterium isotope effect, VH/VD = 8, was observed from pH(pD) 4.2 to 5.4; and at pH(pD) 5.0 the effect was found to be directly related to the mole fraction of 2H. The hydration product, isolated from a beta-amylase/maltal digest in acetate-d4/D2O buffer (pD 5.4) was identified through its 1H NMR spectrum as alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-[2(a)-2H]glucose. beta-Amylase in 2H2O thus catalyzes deuteration of the double bond of maltal from a direction opposite that assumed for protonation of the glycosidic oxygen atoms of starch chains and maltosaccharides. This finding confirms the functional flexibility of the enzyme's catalytic groups first demonstrated in studies of the reactions catalyzed with alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950). A possible mechanism of the maltal hydration by beta-amylase involves protonation of substrate from above as the first and rate-limiting step, followed by formation of a transient carbonium ion-enzyme intermediate. Although other possible mechanisms cannot be ruled out, it is clear that this hydration reaction differs from reactions catalyzed with amylaceous substrates and with alpha- and beta-maltosyl fluoride. The ability of beta-amylase to catalyze different types of reactions with different substrates is discussed with respect to observations with other enzymes that, likewise, strongly support the view (Hehre et al.) that the catalytic groups of glycosylases in general may be functionally flexible beyond requirements of the principle of microscopic reversibility.     

Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain

A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P₄, was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P₄ to the split PH domain-based sensor. The Ins(1,3,4,5)P₄ sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P₄.     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)