Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.     

Mechanism of metabolic regulation in photoassimilation of propionate in Euglena gracilis z

Euglena gracilis showed a typical photoassimilation of propionate when cultured on propionate as a sole carbon source. While the acid is metabolized by the methylmalonyl-coenzyme A (CoA) pathway under illumination, supporting growth of Euglena (K. Hosotani, A. Yokota, Y. Nakano, and S. Kitaoka, 1980, Agr. Biol. Chem.44, 1097–1103), it does not allow the protozoon to grow in the dark although it was actively taken up and metabolized. Kinetics of incorporation of radioactivity of labeled propionate, trapping effect of exogenous lactate in the incorporation of labeled propionate and radiorespirometric pattern revealed that propionate was metabolized by the lactate pathway in Euglena in the dark. Enzymes involved in the lactate pathway were located in mitochondria. The reason why Euglena can not grow on propionate in the dark is explained by the failure of producing C₄ dicarboxylic acids essential for biosynthesis of amino acids and sugars, like the mitochondrial oxidation of fatty acids in higher animals. The Euglena cells cultured in the dark contained enzymes of both methylmalonyl-CoA and lactate pathways, but lack of photosynthetically generated ATP has been suggested to force Euglena to select the less-ATP-requiring but futile pathway.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Growth-related changes in color and sex inHalichoeres poecilopterus

Coloration and sex change were studied in a temperate wrasseHalichoeres poecilopterus in the central part of the Seto Inland Sea, Japan. 1,270 examples, 45–179 mm SL, were collected from May to December both in 1983 and 1984. The species is a diandric, protogynous hermaphrodite, and has three color patterns: pale color type (A), brilliant color type (B) and intermediate color type (AB). A-fish were less than 142 mm SL and consisted of primary males (42.6%), females (55.4%), secondary males (0.3%) and fish with transitional gonads (1.7%). A-females changed their color to B, through AB, in the size range 101–131 mm SL. A-primary males changed their color to B, through AB, in the size range 103–134 mm SL. B-fish consisted of primary males (38.6%), secondary males (54.6%) and fish with transitional gonads (6.8%). The majority of females changed their sex to male in the size range 98–131 mm SL.     

Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA).

Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.     

Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase carrying ribulose 1,5-bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose-1,5-bisphosphate-carboxylase/oxygenase activase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate-binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate-binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246-252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30 degrees C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50 degrees C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0-8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO. The ATP-hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3-5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.