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K Hensley

Publications and source records attributed to K Hensley.

52 records · Page 3Linked to original sources
PubMed1995

beta-Amyloid peptide-derived, oxygen-dependent free radicals inhibit glutamate uptake in cultured astrocytes: implications for Alzheimer's disease.

beta-Amyloid (A beta), the central constituent of senile plaques in Alzheimer's disease (AD) brains, was shown by us recently to generate free radicals in an oxygen dependent mechanism. A beta-derived free radicals were detected directly using electron paramagnetic resonance (EPR) spin trapping techniques employing the spin trap phenyl-alpha-tert-butylnitrone (PBN). We have extended these studies to investigate the nature of the oxyradicals derived from A beta peptides, and we show that these free radicals are able to inhibit glutamate uptake in cultured astrocytes. An implication of inhibited astrocyte glutamate uptake in brain is increased extracellular levels of glutamate, which is excitotoxic to neurons. These results support the hypothesis that A beta neurotoxicity in AD may be due in part to A beta-derived, oxygen-dependent free radical inhibition of glutamate uptake.

Alzheimer Disease↗
PubMed1995

Membrane protein alterations in rodent erythrocytes and synaptosomes due to aging and hyperoxia.

We have applied the technique of electron paramagnetic resonance (EPR) protein-specific spin labeling to the study of membrane protein alterations occurring during age and exposure to isobaric hyperoxia. Cortical synaptosomes and erythrocyte membranes (ghosts) were isolated from young rodents (Fisher 344 rats or mongolian gerbils, 3-4 months of age) and aged rodents (age 22-27 months for rats, greater than 15 months for gerbils). Membrane proteins were spin labeled with the thiol-specific spin label MAL-6 (2,2,6,6,-tetramehtyl-4-maleimido-piperdin-1-oxyl). The relevant EPR spectral parameter of MAL-6 labeled membranes, the W/S ratio, decreased significantly with age of animal in both synaptosomes and ghosts (P < 0.001). As a paradigm for accelerated oxidative stress, young and aged gerbils were exposed to an atmosphere of 90-100% O2 for 0-48 h. In both young and aged gerbils, the W/S ratio decreased significantly with hyperoxic stress (P < 0.003). The W/S ratio of synaptosomes isolated from aged gerbils decreased continually from 0-48 h hyperoxia, whereas the W/S ratio of synaptosomes from young animals demonstrated a pronounced rebound effect from 24-48 h. The results are discussed with reference to membrane protein oxidation in aging.

Aging↗
PubMed1995

Amyloid beta-peptide spin trapping. I: Peptide enzyme toxicity is related to free radical spin trap reactivity.

Synthetic beta-amyloid peptides (A betas) demonstrate lot-to-lot variation in toxicity that has not been adequately explained. Studies from our laboratory have shown that A beta toxicity may result from the ability of the peptide to promote oxidation reactions. Both A beta(1-40) and A beta(25-35) inactivate the oxidation-sensitive enzyme glutamine synthetase (GS) and generate electron paramagnetic resonance (EPR)-detectable products upon reaction with the spin trap phenyl-tert-butylnitrone (PBN). We now report that samples of synthetic A beta(1-40) and A beta(25-35) with attenuated toxicity with respect to peptide-induced GS inactivation, produce qualitatively different EPR spectra when the peptides are incubated with PBN. The results suggest an interpretation of conflicting observations regarding the toxicity of synthetic A betas, and that investigators must be careful to assess the reactivity state of A beta being studied.

Amino Acids↗
PubMed1995

Amyloid beta-peptide spin trapping. II: Evidence for decomposition of the PBN spin adduct.

Synthetic beta-amyloid peptides (A betas) react with the spin trap phenyl-tert-butyl nitrone (PBN) to form products detectable by electron paramagnetic resonance (EPR) spectroscopy. At least two EPR-detectable products can be distinguished from the A beta/PBN reaction, and peptide toxicity towards glutamine synthetase enzyme correlates with the type of PBN reaction product observed. We have reacted synthetic A beta(25-35) peptide with [12C]- or [13C]PBN to demonstrate that the two products represent alternate pathways of spin adduct decomposition. Results indicate that the C=N bond of PBN is cleaved by A beta in what we hypothesize is a radical addition-fragmentation reaction.

Amyloid beta-Peptides↗
PubMed1995

Direct evidence of oxidative injury produced by the Alzheimer's beta-amyloid peptide (1-40) in cultured hippocampal neurons.

The beta-Amyloid peptide (A beta) is hypothesized to mediate the neurodegeneration seen in Alzheimer's disease. Recently, we proposed a new hypothesis to explain the toxicity of A beta based on the free-radical generating capacity of A beta. We have recently demonstrated using electron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) generates free radicals in solution. It was therefore suggested that A beta radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of A beta to produce free-radicals. Using hippocampal neurons in culture, several methods for detection of oxidation were utilized such as the conversion of 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluorescence microscopic method for the detection of carbonyls. The ability of A beta to produce free-radicals was determined using EPR with the spin-trapping compound N-tert-butyl-alpha-phenylnitrone. Consistent with previous studies, we found that preincubation of A beta increased the toxicity of the peptide. There is a strong correlation between the intensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved A beta, which did not generate a detectable radical signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by A beta contributes to the neurodegeneration of Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1995

Enhancement of beta-amyloid peptide A beta(1-40)-mediated neurotoxicity by glutamine synthetase.

The beta-amyloid peptide (A beta), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of A beta(1-40) and its C-end fragment, A beta(25-35). Toxic forms of A beta rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that A beta radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for A beta-derived oxidizing species, the present study was conducted to determine if GS could protect against A beta neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of A beta(1-40). The A beta-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of A beta(1-40) neurotoxicity.

Amyloid beta-Peptides↗
PubMed1995

Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation.

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL-6 spin-labeled synaptosomes, phenylhydrazine-reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)-demented and age-matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation-sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque-dense regions of the AD brain may represent environments of elevated oxidative stress.

Aged↗
PubMed1995

Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death.

The amyloid beta-peptide (A beta) that accumulates as insoluble plaques in the brain in Alzheimer's disease can be directly neurotoxic and can increase neuronal vulnerability to excitotoxic insults. The mechanism of A beta toxicity is unclear but is believed to involve generation of reactive oxygen species (ROS) and loss of calcium homeostasis. We now report that exposure of cultured rat hippocampal neurons to A beta 1-40 or A beta 25-35 causes a selective reduction in Na+/K(+)-ATPase activity which precedes loss of calcium homeostasis and cell degeneration. Na+/K(+)-ATPase activity was reduced within 30 min of exposure to A beta 25-35 and declined to less than 40% of basal level by 3 hr. A beta did not impair other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. Experiments with ouabain, a specific inhibitor of the Na+/K(+)-ATPase, demonstrated that impairment of this enzyme was sufficient to induce an elevation of [Ca2+]i and neuronal injury. Impairment of Na+/K(+)-ATPase activity appeared to be causally involved in the elevation of [Ca2+]i and neurotoxicity since suppression of Na+ influx significantly reduced A beta- and ouabain-induced [Ca2+]i elevation and neuronal death. Neuronal degeneration induced by ouabain appeared to be of an apoptotic form as indicated by nuclear condensation and DNA fragmentation. The antioxidant free radical scavengers vitamin E and propylgallate significantly attenuated A beta-induced impairment of Na+/K(+)-ATPase activity, elevation of [Ca2+]i and neurotoxicity, suggesting a role for ROS. Finally, exposure of synaptosomes from postmortem human hippocampus to A beta resulted in a significant and specific reduction in Na+/K(+)-ATPase and Ca(2+)-ATPase activities, without affecting other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. These data suggest that impairment of ion-motive ATPases may play a role in the pathogenesis of neuronal injury in Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1994

beta-Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer's disease.

We have previously reported (Hensley et al., Proc. Natl. Acad. Sci. USA (1994) in press) that beta-amyloid peptide fragments in aqueous media, in a metal-independent reaction, produce reactive peptide free radicals and reactive oxygen species. In contrast to the hours or days necessary to produce neurotoxicity and a detectable free radical for beta-amyloid, the extremely neurotoxic A beta(25-35) fragment of beta-amyloid peptide produces a detectable radical in minutes. We now report that A beta(25-35) is a potent lipoperoxidation initiator, as inferred from peptide-mediated reduction of nitroxyl stearate spin labels bound to rodent neocortical synaptosomal membranes. A beta(25-35) rapidly quenches the paramagnetism of membrane-bound 12-nitroxyl stearate spin probe deep within the lipid bilayer, but reacts poorly with the 5-nitroxyl isomer whose paramagnetic center is near the lipid/water interface. A beta(35-25), the non-neurotoxic reverse sequence of A beta(25-35), shows little proclivity to reduce either spin label. These findings are formulated into a "molecular shrapnel" model of neuronal membrane damage in Alzheimer's disease.

Alzheimer Disease↗
PubMed1994

A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease.

beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.

Alzheimer Disease↗
PubMed1994

Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure.

Evidence is presented that free radical stress can directly induce physico-chemical alterations in rodent neocortical synaptosomal membrane proteins. Synaptosomes were prepared from gerbil cortical brain tissue and incubated with 3 mM ascorbate and various concentrations of exogenous Fe2+ for 30-240 min at 37 degrees C. Synaptosomes were then lysed and covalently labeled with the protein thiol-selective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperdin-1-oxyl) and subjected to electron paramagnetic resonance (EPR) spectrometry. In separate experiments, synaptosomal membranes were labeled with the thiol-specific spin label MTS ((1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate), or the lipid-specific spin probe 5-NS (5-nitroxide stearate). Free radical stress induced by iron/ascorbate treatment has a rigidizing effect on the protein infrastructure of these membranes, as appraised by EPR analysis of membrane protein-bound spin label, but no change was detected in the lipid component of the membrane. These results are discussed with reference to potential oxidative mechanisms in aging and neurological disorders.

Animals↗
PubMed1993

Alteration of the erythrocyte membrane via enzymatic degradation of ankyrin (band 2.1): subcellular surgery characterized by EPR spectroscopy.

A fraction of band 3 protein, the major transmembrane protein of erythrocyte membranes, is held to the cytoskeletal protein spectrin via noncovalent interactions with the protein ankyrin (band 2.1). In this study, trypsin was used under defined conditions to selectively proteolyze ankyrin and thereby destroy the band 3-ankyrin linkage on the cytoplasmic side of erythrocyte ghost membranes. Electron paramagnetic resonance (EPR) spectroscopy, in conjunction with selective spin labeling methods, was used to monitor conformational changes occurring in cytoskeletal proteins or cell-surface carbohydrates as a result of this treatment. Treatment of RBC ghosts with TPCK-trypsin for 5 s at 0 degrees C caused an approx. 56% increase in the relevant EPR parameter of a maleimide spin label bound to spectrin (P < 0.004), indicative of increased segmental motion of the spin label and decreased protein-protein interactions. Analysis of the apparent rotational correlation time parameter tau of a spin label covalently and selectively bound to terminal sialic acid residues of glycophorin showed no significant effect from trypsin treatment. However, tau of spin label covalently and specifically bound to terminal galactose residues of cell-surface glycoconjugates of band 3 and other transmembrane glycoproteins significantly decreased with tryptic uncoupling of the ankyrin linkage (P < 0.005). These results suggest a marked conformational alteration in both cytoskeletal and transmembrane proteins as a result of uncoupling from ankyrin. Spermine (N,N'-bis(3-aminopropyl)tetramethylenediamine), a naturally occurring polyamine known to strengthen cytoskeletal protein-protein interactions (Wyse and Butterfield (1988) Biochim. Biophys. Acta 941, 141-149), was used to partially reverse the trypsin-induced cytoskeletal alterations. Addition of 2 mM spermine to ghosts previously treated with trypsin increased cytoskeletal protein-protein interactions as indicated by EPR (P < 0.002). SDS-PAGE was used to confirm the integrity of spectrin, band 3, and band 4.1 in all experiments. The results are discussed with reference to transmembrane signaling mechanisms and membrane-associated pathologies.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1993

Interaction of tacrine and velnacrine with neocortical synaptosomal membranes: relevance to Alzheimer's disease.

The acridine-based, potential Alzheimer's disease therapeutic agents, tacrine and velnacrine, were incubated with rat or gerbil neocortical synaptosomal membranes. Electron paramagnetic resonance employing a protein-specific spin label was used to monitor this interaction. Analogous to their effects in erythrocyte membranes [Butterfield and Rangachari (1992) Biochem. Biophys. Res. Commun. 185: 596-603], in the present studies both agents decreased segmental motion of spin labeled synaptosomal membrane proteins, consistent with increased cytoskeletal protein-protein interactions (0.001 < P < 0.005), and tacrine was more potent than velnacrine. These results are discussed with possible relevance to molecular actions of the agents and molecular alterations in Alzheimer's disease.

Alzheimer Disease↗
PubMed1993

Breast cancer in Florida women. Incidence and stage at diagnosis.

Using the Florida Cancer Data System, trends were examined in female breast cancer incidence and stage of diagnosis over the period 1981-1989. The crude incidence rate increased 20% due to diagnosis at local stage or in situ. Incidence rates of disease with regional or distant spread have declined. Similar trends appear in all age groups statewide and in both Hispanic and non-Hispanic whites. The shift to earlier stage at diagnosis is not seen for nonwhite women who are less likely to be diagnosed at a local stage than white women, even after controlling for the younger age distribution of nonwhite cases. For non-Hispanic whites, the percent local at diagnosis is lowest in women in urban counties. Careful breast examination and mammographic screening for asymptomatic women lead to earlier diagnosis, longer survival and even cure. While considerable progress has been made in early detection, nonwhites and some urban residents in Florida have not received the full benefit of these changes.

Adult↗
PubMed1993

Breast cancer screening in Florida. Opportunities for prevention.

Records of 705 women age 40 years and over interviewed by the Florida Behavioral Risk Factor Surveillance System in 1990 were used to identify gaps in mammography utilization in Florida. Data were weighted to the 1990 age and race composition of the population. Of the women 55.8% received a mammogram in the last two years and 33.4% reported they had never had one. The percentage reporting a mammogram in the last two years was 61.9% for those who had a check-up in the last year and 68.7% for those who had a breast examination in the last year. The reason 34% gave for not having a mammogram was "not recommended by doctor." Cost or lack of insurance was the reason for 10.6% of all women and 15.2% of women from households below $15,000 annual income. Data were further stratified by age and race/ethnicity. Significant missed opportunities for mammography are occurring among women in the health-care system. Interventions to increase mammography need to address providers' attitudes and behaviors as well as potential patients.

Adult↗