Myocardial uptake kinetics of tocainide enantiomers in the isolated perfused rabbit heart

The extent of myocardial accumulation of tocainide, administered as single enantiomers and as well as racemate, was determined in the isolated, spontaneous beating rabbit heart. The heart was retrogradely perfused at a constant rate and fractions of the perfusate were collected during and after infusion. Kinetic parameters for myocardial accumulation and disposition of tocainide were indirectly determined from drug concentration/time course in the outflow perfusate. No stereoselectivity in myocardial accumulation was observed. A two compartment model with mean half-lives for distribution and elimination of 0.60 and 3.78 min, respectively, was fitted to the accumulation and disposition data. At steady-state, tocainide enantiomers were accumulated about three times in the myocardium relative to the perfusion liquid. © 1995 Wiley-Liss, Inc.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

Further studies on the subcellular localization of lipid-degrading enzymes

Further work on the subcellular localization of two lipid-degrading enzymes, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX) has been carried out on brassica florets, potato shoots and pea roots. In all cases, the LAH profile on sucrose and Ficoll density gradients was coincident with ‘lysosomal’ acid phosphatase activity. However, the localization of LOX activity was different for each tissue. In pea roots the activity of LOX was localized in the ‘lysosomal’ fraction, whereas with brassica florets (cauliflower and calabrese) it was present in a heavy body with a similar density to plastids and in potato shoots LOX gave only low particulate recoveries.     

Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication.

The number of proline residues in a protein should have very marked consequences for the rates of protein unfolding and refolding according to the model proposed by Brandts et al. (1975). Kinetic simulations of this model indicate that the half-time for refolding of a polypeptide chain with 20 proline residues should be greater than 10 minutes and should increase by about an order of magnitude for each additional 10 proline residues. Various means are considered by which the rate of protein folding in vivo and in vitro might be increased.     

Generation of formic acid and ethanolamine from serine in biosynthesis of linear gramicidin by a cell-free preparation of Bacillusbrevis (ATCC 8185)

A growing organism that produces antibiotic peptide was incubated with L-(U-¹⁴C)serine for labeling linear gramicidin. Linear gramicidin was isolated by a simple chromatographic method from tyrothricin (mixture of linear gramicidin and tyrocidine) applied to a column of basic aluminum oxide. The hydrolysate of labeled linear gramicidin on thin layer chromatography showed that L-(U-¹⁴C)serine was one of a precursor of ethanolamine moiety by autoradiography. L-(3-¹⁴C)serine generated formic acid in the presence of tetrahydrofolic acid by an enzyme fraction prepared with ammonium sulfate, and further formed ethanolamine binding to the protein. Formylvaline was biosynthesized by it with tetrahydrofolic acid and ATP, and subsequently released from the protein.     

MIXED MODEL APPROACHES FOR ESTIMATING GENETIC VARIANCES AND COVARIANCES

The limitations of methods for analysis of variance(ANOVA)in estimating genetic variances are discussed. Among the three methods(maximum likelihood ML, restricted maximum likelihood REML, and minimum norm quadratic unbiased estimation MINQUE)for mixed linear models, MINQUE method is presented with formulae for estimating variance components and covariances components and for predicting genetic effects. Several genetic models, which cannot be appropriately analyzed by ANOVA methods, are introduced in forms of mixed linear models. Genetic models with independent random effects can be analyzed by MINQUE(1)method whieh is a MINQUE method with all prior values setting 1. MINQUE(1)method can give unbiased estimation for variance components and covariance components, and linear unbiased prediction (LUP) for genetic effects. There are more complicate genetic models for plant seeds which involve correlated random effects. MINQUE(0/1)method, which is a MINQUE method with all prior covariances setting 0 and all prior variances setting 1, is suitable for estimating variance and covariance components in these models. Mixed model approaches have advantage over ANOVA methods for the capacity of analyzing unbalanced data and complicated models. Some problems about estimation and hypothesis test by MINQUE method are discussed.