Richard A. Haak

Lactoferrin Deficiency Associated with Altered Granulocyte Function

EIGHT years ago Strauss et al. reported unique structural abnormalities in the granulocytes of a boy who had had recurrent bacterial infections since birth.1 The nuclei were bilobed, and in ultrast...

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.

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.

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.

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.

THE INFLUENCE OF VITAMIN E ON HUMAN POLYMORPHONUCLEAR CELL METABOLISM AND FUNCTION

These studies on the effect of administration of 1,600 units of vitamin E to humans indicated the following responses to the PMNs (TABLE 6). Functional alterations occur with an increased ability to ingest particles but a mild decrease in bactericidal potency of the PMN. Although the respiratory burst is slightly enhanced as is superoxide anion release, H2O2 release from the PMN is markedly impaired. The hexose monophosphate shunt activity, which is dependent on intracellular H2O2 is decreased during phagocytosis. Membrane responses such as changes in order parameter during phagocytosis as reported by the stearic acid analogue probe 5DS are similar to those of normal PMNs. The release of arachidonic acid from membranes of vitamin E PMNs during phagocytosis of opsonized zymosan is slightly enhanced, indicating normal phospholipase A2 activation. NADH oxidase-derived H2O2 is not impaired within phagocytic generated by NADPH oxidase in phagocytic vesicles, accounting for impairment in HMPS activity and bactericidal activity in these cells.

Electron spin resonance spectroscopy does not reveal hydroxyl radical production in activated natural killer lymphocytes

We investigated hydroxyl radical (OH) production by human natural killer (NK) cells, using electron spin resonance (ESR) spectroscopy and 5,5 dimethyl‐1‐pyrroline‐N‐oxide (DMPO), a spin trap specific for OH production. We confirmed that hydroxyl radical scavengers, n‐propyl gallate and catechin, inhibited NK cell‐mediated cytotoxicity (NK‐CMC) in a dose‐dependent manner and demonstrated that DMPO also inhibited NK‐CMC. Polymorphonuclear leukocytes (PMNL) activated by opsonized zymosan (2.4 mg/ml) and mixed with DMPO (0.14 M) showed an early increase in hydroxyl radical production, leading to a net production of free radical of almost 400 pMol/106 cells. We then mixed NK cells with K562, an NK‐sensitive tumor cell, at a 1:1 ratio and added DMPO (0.14 M). We pelleted the cells to increase EC to TC binding before taking the sample readings. Activated NK cells showed no increase in OH production, leading to a net production of free radicals less than 1% that of activated PMNL. These data strongly suggest that hydroxyl radical production does not play a role in the early events of NK cell activation; they indicate a need to reevaluate the mechanism of inhibition of NK‐CMC by OH scavengers.

Effect of vitamin E on FMLP-induced activation of rabbit polymorphonuclear leukocytes

Granulocytes of vitamin E-treated rabbits were compared to granulocytes from placebo-treated rabbits. Granulocytes were isolated from rabbit peripheral blood by a new method employing Percoll and gelatin sedimentation. Vitamin E-treated cells showed less adherence to rabbit aortic endothelium when stimulated with FMLP. FMLP receptor numbers and affinity were not significantly different. Resting cell surface and baseline transmembrane potential were similar in both cell types. Decrease in cell surface potential with FMLP was comparable in vitamin E- and placebo-treated cells. Vitamin E-treated PMN depolarized more and hyperpolarized more rapidly than placebo cells. Thus vitamin E-treated PMNs show differences in the early events of PMN activation. These may contribute to the lower stimulated adherence observed with vitamin E-treated cells.

Cultured Cells as Model Systems in Shock Wave Lithotripsy Research: Advantages, Methodological Concerns and Potential Applications

Shock waves generated during extracorporeal shock wave lithotripsy (ESWL†) therapy have produced both renal and extrarenal tissue injury.1–4 Renal injury, although not yet fully characterized, includes disruption of the vasculature, damage to the tubular epithelium, and apparent alterations in the renal interstitium. Whole animal studies are being used to document the types of tissue injury that occur during ESWL and are suitable for the investigation of how acute injury may lead to chronic alterations in kidney structure and function. However, in vivo studies may neither allow the adequate probing of the cellular basis of ESWL injury nor aid the understanding of the mechanism by which cells of the kidney are damaged. Such studies cannot precisely determine what role the various physical parameters of shock wave delivery play in producing cell injury. Therefore, investigators have employed cultured cells as model systems to investigate how shock waves injure cells and to determine the role of physical parameters in producing cell/tissue injury. A rationale for employing cultured cells as model systems in ESWL research is presented here, along with a discussion, from a tissue culture perspective, of the advantages and limitations of using isolated cells to better understand how shock waves injure cells of the intact kidney.