Purification and immunological characterization of superoxide dismutase of the onion maggot,Delia antiqua

Cytosolic superoxide dismutase (SOD) of the onion maggot, Delia antiqua, was purified to apparent homogeneity by ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and gel filtration chromatographies. Native molecular mass was estimated as 32,000 daltons. SDS-PAGE revealed only one subunit of 16,000 daltons, indicating that SOD is a homodimer. Isoelectric focusing revealed 3 charge isomers of pls 5.3, 5.5, and 5.7. The specific activity of purified SOD was 4,250 U/mg protein. A monoclonal antibody (MAb, aSOD2B7) raised against Delia SOD recognized only SOD of the same genus, but another MAb (aSOD1H11) recognized SOD of Drosophila melanogaster as well. © 1995 Wiley-Liss, Inc.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

The role of ankyrin in shape and deformability change of human erythrocyte ghosts

Human erythrocyte membranes (ghosts) from acid/citrate/dextrose preserved blood were digested with trypsin (protein/trypsin = 100:1) under hypotonic conditions and then analyzed by SDS-polyacrylamide gel electrophoresis. After digestion for about 20-30 s at 0 degree C, only ankyrin had disappeared and other bands including spectrin, actin, band 4.1 and band 3 remained intact. This observation was supported by electron micrographs showing that the horizontally disposed, filamentous structure was a little apart from the lipid bilayer and its components were not destroyed. In contrast to intact ghosts, treatment with chlorpromazine, or Mg-ATP did not induce shape change in these trypsin-treated ghosts. The number of transformable cells correlated closely with the amount of remaining ankyrin in the SDS-polyacrylamide gel electrophoresis pattern. Furthermore, the chlorpromazine- and Mg-ATP-induced decreases in viscosity of suspensions of erythrocyte ghosts were also prevented by trypsin treatment for 20-30 s at 0 degree C. These findings suggest that ankyrin plays an important role in the change in shape and deformability of erythrocyte ghosts. The molecular mechanism of drug-induced shape change and the role of undermembrane structure in regulating erythrocyte shape and deformability are discussed.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Dielectric properties of spherical macroion suspensions. I. Study on monodisperse polystyrene latex

Dielectric properties of polystyrene latex suspended in aqueous media are investigated with special attention to the effect of volume fraction of the latex and salt concentration. The experimental data show the existence of two dispersions, one in the low-frequency range from 10(3) to 10(5) Hz. and the other in the high-frequcncy range from 10(5) to 10(7) Hz. In the salt-free system. both dispersions are of the Debye type and their relaxation limes arc insensitive to the volume fraction. Addition of H2SO4 to the suspension enlarges the magnitude of the low-frequency dispersion and reduces that of the high-frequency dispersion. whereas it does not affect the relaxation times. In the mixture of two species of counterions, e.g. H+ and Na(+), the low-frequency dispersion deviates from the Debye type. while the high-frequency dispersion docs not. From these facts, the high- and low-frequency dispersions are thought to be due to the radial and tangential components of the displacement current at the surface of the latex. The latter process is consistent with the Schwarz theory of the dielectric dispersion of colloidal suspensions.     

Chronotropism and blood flow patterns following teratogenic doses of catecholamines in 5-day-old chick embryos.

In vivo heart rates of 5-day-old chick embryos were recorded from electrodes placed in close proximity to the heart. L-epinephrine (4X10(-10) mole), 1-norepinephrine (1X10(-9)mole) and 1-isoproterenol (1.6X10(-10)mole) in 5 microliter of isotonic saline transiently accerlerated the mean heart rate by almost 9 percent. L-phenylephrine (2X10(-9)mole/5microliter) and the experimental procedure produced no appreciable effect. The positive chronotropic effect of the catecholamines was found to be highly significant (P less than 0.0005) as computed by Student's t test. However, no direct relationship could be established between the chronotropic response and the aortic arch anomalies produced. A prolonged reduction of blood flow in the primitive heart tube and the sixth aortic arch after administration of epinephrine and isoproterenol is apparently related to the induction of hypoplastic right pulmonary artery with absent or hypoplastic right ductus arteriosus.