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Biomedical subjects

R A Haak

Publications and source records attributed to R A Haak.

30 records · Page 2Linked to original sources
PubMed1980

Resolution of basic gonococcal outer membrane proteins by nonequilibrium pH gradient electrophoresis.

Outer membrane proteins from opaque and transparent colonial variants of strain F62 of Neisseria gonorrhoeae were analyzed by two-dimensional electrophoresis with isoelectric focusing in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. Most of the higher-molecular-weight proteins focused sharply in the acidic region of the gel. In contrast, the principal outer membrane protein, a 31,000-molecular-weight protein, and the opacity-associated proteins remained near the origin (at the basic end of the gel) without focusing. However, when the samples were loaded on the acidic end of an isoelectric focusing gel and subjected to nonequilibrium pH gradient electrophoresis, these proteins behaved as basic proteins. In addition, three distinct opacity-associated heat-modifiable proteins could be identified. No other differences in the protein composition of outer membranes from opaque and transparent variants were apparent. Amino acid analysis of the principal outer membrane protein indicated that its net positive charge may result from partial amidation of its acidic residues. The unexpected observation that the major surface proteins of the gonococcus are basic may have implications for intragonococcal adhesion and for gonococcal interactions with mammalian cells.

Amino Acids↗
PubMed1979

ESR determination of membrane order parameter in yeast sterol mutants.

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.

Cell Membrane↗
PubMed1979

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.

Cell Membrane↗
PubMed1979

Membrane fluidity in human and mouse Chediak-Higashi leukocytes.

Polymorphonuclear leukocytes from humans and mice with the Chediak-Higashi syndrome were characterized by spin label electron spin resonance spectrometry. Our results suggest that cells from afflicted mice and humans have membranes more fluid than controls. Order parameters for a spin label that probes near the membrane surface were 0.652 for normals and 0.645 for two Chediak-Higashi patients. Cells from Chediak-Higashi mice showed similar differences, as did isolated plasma membrane fractions. An increased membrane fluidity was also detected with a spin label that probes deeper in the bilayer. In vitro treatment of Chediak-Higashi mouse cells with 0.01 M ascorbate increased the order parameter to normal levels. In vitro incubation of mouse Chediak-Higashi cells with glucose oxidase increased the order parameter, similar to the effect of ascorbate. This increase was abolished when catalase was added to the incubation medium. In vitro incubation with dibutyryl cyclic guanosine monophosphate (1 muM to 0.1 mM) did not normalize order parameters. These results indicate that fluidity of Chediak-Higashi cell membranes was affected by treatments expected to alter the oxidation: reduction potential of the environment but was not affected by treatments expected to alter the ratio of intracellular cyclic nucleotides. The latter treatment would affect microtubule assembly. Therefore, it appears that the membrane fluidity abnormalities as demonstrated by electron spin resonance and the earlier demonstrated microtubule dysfunctions characteristic of Chediak-Higashi cells are coexisting defects and are probably not directly related.

Animals↗
PubMed1976

The viscosity of mammalian nerve axoplasm measured by electron spin resonance.

1. The microviscosity of the axoplasm of can sciatic nerve was determined by an in vitro electron spin resonance (e.s.r.) method using the spin label tempone. To identify the spin label signal as one arising only from within the axoplasm, Ni2+ was used as a line broadening agent. In one series of experiments in nerves with sheath intact the Ni2+ ion was shown to eliminate the tempone signal arising from the surface water, and in another series of experiments, with the sheath slit, to eliminate the signal from the extracellular space as well. 2. A microviscosity of less than 5 centipoise (cP), i.e. 5x that of water, was determined for the axoplasm. Changes in the viscosity of the nerve axoplasm as a function of temperature over a range of 38 degrees down to 2 degrees C were seen to follow closely the viscosity change found for a water solution. 3. The microviscosity of nerve axoplasm and its change with temperature were related to axoplasmic transport of material in nerve fibres. The results were used to exclude a large increase in viscosity at low temperatures as the cause for the cold-block of fast axoplasmic transport.

Animals↗
PubMed1975

Plasmid DNA in virulent and avirulent gonococci.

The presence of covalently closed circular (CCC) DNA in virulent and avirulent colonial types of Neisseria gonorrhoeae strain 2686, and of type 3 colonies of strain F62 was investigated. CCC DNA was present in each, and the percentage of plasmid relative to total DNA was similar. The percentage of CCC DNA was similar in cells grown on a solid as compared with a liquid medium and in cells grown in the presence of varying concentrations of glucose. No correlation could be established between the presence of plasmid DNA and virulence of N. gonorrhoeae.

Culture Media↗
PubMedDate not supplied

ESR determinations of membrane permeability in a yeast sterol mutant.

Yeast sterol mutants were subjected to ESR analysis in an attempt to elucidate how altered sterol composition correlates with membrane permeability. The technique requires spin labeling the intact yeast cells with a small, water-soluble nitroxide probe (2,2,5,5 tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid, PCA), suspending cells in a NiCl2 solution, and measuring the extent of Ni2+ entry through the membrane by its magnetic dipolar line broadening effect on the PCA signal. The wild type, A184D, was found to be impermeable to Ni2+ during all growth phases while the sterol mutant erg 6/2 was readily permeable to Ni2+. Other sources of line broadening such as increased rotational correlation time and cell nonviability are shown to be neglibible. Internal Ni2+ concentrations for erg 6/2 and kinetics of Ni2+ entry were determined.

Cell Membrane Permeability↗
PubMedDate not supplied

Psychostimulant effects on neuropsychological, intellectual, and achievement measures for children and adolescents with Attention Deficit Hyperactivity Disorder.

This study compared the performance of medicated (n=57) and non-medicated (n=20) subjects with Attention Deficit Hyperactivity Disorder (ADHD) on neuropsychological, intellectual, and achievement measures. The Wechsler Intelligence Scale for Children (Revised or 3rd Edition), appropriate Halstead Reitan Neuropsychological Test Battery, and individual achievement tests were administered to subjects between the ages of 9 and 16 years (M=12.8) Initially, 26 subtest scores were analyzed using multivariate profile analysis. The two groups were found to be parallel (i e, similar in pattern of performance) and coincident (i.e., similar in level of performance). This suggests that the subjects in the two groups displayed a similar pattern of performance on these neuropsychological, intellectual, and achievement measures. The results are discussed in relation to previous research, current etiological models, and the assessment and treatment of ADHD.

Journal Article↗