PubMed Health⌕ Search

Biomedical subjects

A Keith

Publications and source records attributed to A Keith.

At least 19 recordsLinked to original sources
PubMed1984

Alteration by prolactin of surface charge and membrane fluidity of rat 13762 mammary ascites tumor cells.

Intraperitoneal injection of ovine prolactin (100 micrograms/d) in Fischer 344 rats bearing transplantable 13762 mammary ascites tumor (MAT) cells modifies the surface charge density and membrane fluidity of the tumor cells. In each of five experiments the mean electrophoretic mobility (epm) of MAT cells taken from prolactin-treated rats was significantly lower than that of cells from nonhormone-treated controls. Prolactin concentrations were increased in vivo by (a) direct intraperitoneal injection of ovine prolactin; (b) subcutaneous implantation of diethylstilbestrol-containing silastic capsules to produce pituitary prolactin secreting tumors; or (c) a single subcutaneous injection of polyestradiol phosphate, a long-acting estrogen. In an effort to establish that the prolactin effect was a direct one, two in vivo protocols were used: (a) MAT cells were coincubated with anterior pituitary halves obtained from nontumor-bearing littermates; or (b) rat or ovine prolactin was added to serum-free culture media containing MAT cells. In both protocols, the epm of the prolactin-treated cells was significantly lower. The isoelectric focusing pH of whole cells was increased by prolactin treatment from 4.93 to 5.12, consistent with a reduction in the number of surface carboxyl groups. The fluidity of membranes of treated cells was drastically increased, as measured by spin-label probe rotation rates. These combined results imply that the hormone exerts its effect by stimulating events in the cell that lead to a reduction of the average density of carboxylic acid residues on the tumor cell surface.

Animals↗
PubMed1980

Interaction of divalent cations and proteins with phospholipid vesicles.

The broadening of spin-label absorption lines resulting from spin-exchange reactions that occur during collision with paramagnetic Ni2+ is diminished when Ni2+ binds to phospholipid vesicles. Subsequent addition of non-paramagnetic ions that compete for binding sites releases Ni2+ into solution and restores the line-broadening. The concentrations of various ions required to achieve this effect was used to order the ions with respect to their binding to vesicles containing phosphatidylethanolamine and phosphatidylglycerol. The relative strengths of binding for those ions studied were: Ca2+ > Mg2+ > Zn2+ > Sr2+ > Ba2+. The spin-broadening assay was also used to study the effects of two proteins on the availability of Ni2+-binding sites on the vesicles. Ribonuclease, which is thought to associate electrostatically as an extrinsic protein on the surface of vesicles, completely blocked the Ni2+-binding sites at comparatively low protein concentrations. Quantitative considerations of these data suggest the possibility that Ni2+ may bind preferenetially to phosphatidylglycerol, and that these binding sites are aggregated in the ribonuclease-containing vesicles. In contract to ribonuclease, cytochrome c does not block Ni2+-bindings sites on the phospholipid vesicles, but rather contains sites of its own that bind Ni2+, both when the protein is in solution and when it is associated with the vesicles. These results are consistent with other studies which suggest that cytochrome c becomes partially embedded in membrane bilayers and associates with phospholipid molecules through hydrophobic interactions.

Binding Sites↗
PubMed1980

Studies with a hydrophobic, spin-labeled virucidal agent.

A spin-labeled virucidal agent was synthesized, purified, and tested for its activity against the enveloped bacterial virus phi6 and herpes simplex virus. This compound, designated BPN, inactivated greater than 99% of phi6 and herpes simplex virus in vitro at concentrations as low as 0.1 mM. Detailed studies were carried out on the mechanism of inactivation of phi6 by BPN. These studies revealed that treatment of phi6 by BPN specifically removes a single envelope protein that is considered to be responsible for adsorption of the virus to the host cell. Related experiments with the phi6 host, Pseudomonas phaseolicola strain HB10Y, showed that this organism is insensitive to the effects of BPN. The basis for the differential sensitivity of phi6 to BPN, in comparison to the host cell, was investigated by electron spin resonance techniques. It was found that, for phi6, HB10Y, and their extracted phospholipids, BPN is localized in the hydrocarbon zones of the membrane bilayer. However, in the case of phi6, the rotational mobility of BPN is much reduced in comparison to that in HB10Y and the phospholipid preparations. Furthermore, an Arrhenius plot of rotational correlation time (tau(c)) showed a marked discontinuity in slope at 31 degrees C in the case of phi6, but not for the other samples studied. This suggests a strong interaction between the phi6 envelope proteins and the lipid domains in which BPN is localized. Calculations based on the known lipid and protein composition of phi6 indicate that there is an absence of "free-lipid" pools in the viral envelope. It is suggested that BPN localizes in free-lipid pools of cell membranes, where its presence is of little or no consequence, but that in phi6 the BPN perturbs the hydrophobic interactions between phospholipids and proteins in the envelope.

Antiviral Agents↗
PubMed1977

Inactivation of lipid-containing viruses by long-chain alcohols.

This report describes the inactivation of lipid-containing viruses by several long-chain alcohols. A striking peak in antiviral activity was found for saturated alcohols having chain lengths from 10 to 14 carbons. Viruses having different membrane structure showed different susceptibilities to alcohols having different chain lengths and structural features. Decanol, dodecanol, and tetradecanol readily inactivated herpes simplex virus and the enveloped bacterial virus phi6. The lipid-containing virus PM2 was susceptible to decanol and dodecanol but comparatively unsusceptible to tetradecanol. The branched-chain alcohol phytol, a naturally occurring component of chlorophyll, was active against phi6 and herpes simplex virus but not against PM2. Polyoma virus and the bacteriophage phi23-1-a, which do not contain lipids, were not susceptible to inactivation by any of the alcohols tested. Experiments were also carried out to determine the effects of these compounds on cells. At 0.5 mM, decanol lysed human embryonic lung cells, erythrocytes, and the bacterial hosts for phi6 and PM2. Dodecanol, tetradecanol, and phytol at this concentration were less damaging to cells. At 0.05 mM, none of the alcohols caused observable cytopathic effects on human embryonic lung cells, although several of the alcohols at this concentration were active against herpes simplex virus. Our findings suggest that dodecanol, tetradecanol, and phytol may warrant further studies as potential antiviral agents, particularly for topical application to virus-infected areas of the skin.

Antiviral Agents↗
PubMed1975

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.

Anti-Bacterial Agents↗
PubMed1975

Inactivation of the lipid-containing bacteriophage PM2 by butylate hydroxytoluene.

Several factors have been investigated which are of significance in the inactivation of PM2, a lipid-containing bacterial virus, by butylated hydroxytoluene (BHT). Studies of the time dependence of inactivation during exposure to BHT showed that virus killing occurs rapidly, with the majority of the effect taking place in the first 5 min. The degree of inactivation is dependent upon the initial virus titer, the solvent from which BHT is added, and the presence of a variety of protective agents, including surfactants, bovine serum albumin, and bacterial cells. Sucrose gradient analysis of (32)P-labeled, BHT-treated virus was used to determine the degree to which the virion is disrupted by BHT. These experiments show that the (32)P-labeled molecules are converted into very slowly sedimentable material by BHT treatment, indicating complete destruction of the virus particle.

Bacteriophages↗
PubMed1974

Electron spinal resonance analysis of the nitroxide spin label 2,2,6,6-tetramethylpipidone-N-oxyl (Tempone) in single crystals of the reduced Tempone matrix.

The nitroxide spin label Tempone (2,2,6,6-tetramethylpiperidone-N-oxyl) can be reduced with ascorbic acid to give a nonparamagnetic species. Single crystals of reduced Tempone serve as a suitable host matrix to orient trace quantities of Tempone for ESR analysis. In these crystals the majority of the Tempone molecules are well-oriented, but a smaller fraction of the molecules tumble freely to give an isotropic electron spin resonance (ESR) spectrum. ESR transitions for the oriented molecules are saturated at much lower microwave power levels than for the tumbling molecules. For the oriented molecules, an analysis of the anisotropy of the spectroscopic splitting factor (g) gives principal values of g(1) = 2.0094, g(2) = 2.0061, g(3) = 2.0021. The hyperfine coupling tensor is nearly axially symmetric, with principal values (in gauss) of A(1) = 6.5, A(2) = 6.7, A(3) = 33.0. Within experimental error, the principal axis systems for the g tensor and the hyperfine tensor are identical. Comparison of the average values of g and A with the isotropic values of these parameters for Tempone in solvents of different polarity suggests a method for choosing the most appropriate tensor elements to be used for spin label experiments in various solvent systems.

Crystallization↗
PubMed1974

Active transport of choline by a marine pseudomonad.

A marine pseudomonad, BAL-31, accumulates the phospholipid nitrogen base, choline, although no detectable amount of choline is incorporated into polar lipids. Metabolic inhibitors such as cyanide and azide block the uptake process as does starving for oxygen by using nitrogen gas. Only very close structural analogues show any inhibition of transport, indicating that the uptake process has great structural specificity. The export of choline out of the cells is also an energy-dependent process and is markedly reduced during oxygen depletion. The constitutive level of choline transport is increased by approximately a factor of three after a brief induction period. Two other gram-negative bacteria also accumulate choline, whereas a gram-positive bacterium, Bacillus subtilis, and a yeast, Saccharomyces cerevisiae, fail to show any detectable accumulation.

Acetylcholine↗
PubMed1973

Spin label motion in fatty acids.

Spin labels dissolved in highly purified fatty acid systems exhibit nearly identical tumbling rates in liquid and solid phases. Even though the spin labels do not have the same molecular geometry as the lipid matrix the melting point of the matrix can be inferred by measurements of the temperature dependency of molecular motion.

Chromatography, Thin Layer↗