Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.     

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.     

The plasma membrane of myxosporidian valve cells: freeze fracture data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.     

Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions

We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.     

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.     

Transcobalamin II--cobalamin binding sites are present on rabbit germ cells.

Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.     

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.