D1-D2-cytochrome b559 complex from the aquatic plant Spirodela oligorrhiza: correlation between complex integrity, spectroscopic properties, photochemical activity, and pigment composition

A D1-D2-cyt b559 complex with about four attached chlorophylls and two pheophytins has been isolated from photosystem II of the aquatic plant Spirodela oligorrhiza and used for studying the detergent-induced changes in spectroscopic properties and photochemical activity. Spectral analyses (absorption, CD, and fluorescence) of D1-D2-cyt b559 preparations that were incubated with different concentrations of the detergent Triton X-100 indicate two forms of the D1-D2-cyt b559 complexes. One of them is photochemically active and has an absorption maximum at 676 nm, weak fluorescence at 685 nm, and a strong CD signal. The other is photochemically inactive, with an absorption maximum at 670 nm, strong fluorescence at 679 nm, and much weaker CD. The relative concentrations of the two forms determine the overall spectra of the D1-D2-cyt b559 preparation and can be deduced from the wavelength of the lowest energy absorption band: preparations having maximum absorption at 674, 672, or 670.5 nm have approximately 20, 60, or 85% inactive complexes. The active form contains two chlorophylls with maximum absorption at 679 nm and CD signals at 679 (+) and 669 nm (-). These chlorophylls make a special pair that is identified as the primary electron donor P-680. The calculated separation between the centers of these two pigments (using an extended version of the exciton theory) is about 10 A, the pigments' molecular planes are tilted by about 20 degrees, and their N1-N3 axes are rotated by 150 degrees relative to each other. The other two chlorophylls and one of the two pheophytins in the D1-D2-cyt b559 complex have their maximum absorption at 672 nm, while the maximum absorption of the photochemically active pheophytin is probably at 672-676 nm. During incubation with Triton X-100, the photochemically active complex is transformed into an inactive form with first-order kinetics. In the inactive form the maximum absorption of the 679 nm absorbing Chls is blue-shifted to 669 nm. The first-order decay of the photochemical activity suggests that the isolated D1-D2-cyt b559 complex is stable as an aggregate but becomes unstable on dissociation into individual D1-D2-cyt b559 units.     

Cloning of the aspartate-β-semialdehyde dehydrogenase structural gene fromEscherichia coli K12

Transformation ofEscherichia coli strains with the recombinant plasmid, prepared by shot gun cloning with pBR322 and containing the geneasd, the structural gene of aspartate--semialdehyde dehydrogenase, results in an increase in specific activity of 65-fold of the enzyme in crude extracts. Approximately 60 mg of pure enzyme may be obtained from 10 g of transformed cells (wet weight) in a simplified purification procedure. The molecular weight, amino acid composition, and kinetic properties of the enzyme appear to be the same as previously reported, and the first 36 amino acids of the N-terminal sequence have been determined.     

Liquid chromatographic determination of caffeine in biologic fluids

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 μg/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.     

Murine transfer factor. I. Description of the model and evidence for specificity

A model for studying transfer of delayed-type sensitivity to mice with cellfree materials is described. The results with a particulate antigen (Candida) and 4 soluble protein antigens (PPD, ferritin, cytochrome c, and horseradish peroxidase) suggest that the phenomenon is antigen specific. Identical preparations from the spleens of insensitive donors were not active. This murine model should facilitate characterization of the immunologic and chemical properties of transfer factor.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Role of collagen and fibronectin in neural crest cell adhesion and migration

Cultured neural crest cells which are freshly trypsinized require serum or purified fibronectin to attach to collagen substrates of types I–V. Furthermore, neural crest cells migrate in a Boyden chamber in response to fibronectin, and a “checkerboard” analysis demonstrates that fibronectin is both chemotactic and chemokinetic for these cells. It is proposed that collagen serves as a substrate for neural crest cells as they migrate in the early embryo. By mediating the cells' attachment to collagen, fibronectin may influence the movement of the cells. Local differences in fibronectin concentration or availability in the matrix could affect the degree of attachment of the cells to the collagen substrate and could also direct their migration by serving as a chemoattractant.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.