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Biomedical subjects

James P Bennett

Publications and source records attributed to James P Bennett.

At least 19 recordsLinked to original sources

Factors to consider for trace element deposition biomonitoring surveys with lichen transplants.

A trace element deposition biomonitoring experiment with transplants of the fruticose lichen Evernia prunastri was developed, aimed at monitoring the effects of different exposure parameters (exposure orientation and direct rain) and to the elements Ti, V, Cr, Co, Cu, Zn, Rb, Cd, Sb and Pb. Accumulations were observed for most of the elements, confirming the ability of Evernia transplants for atmospheric metal deposition monitoring. The accumulation trends were mainly affected by the exposure orientation and slightly less so by the protection from rain. The zonation of the trace elements inside the thallus was also studied. It was concluded that trace element concentrations were not homogeneous in Evernia, thus imposing some cautions on the sampling approach. A nuclear microprobe analysis of an E. prunastri transplanted thallus in thin cross-sections concluded that the trace elements were mainly concentrated on the cortex of the thallus, except Zn, Ca and K which were also present in the internal layers. The size of the particles deposited or entrapped on the cortex surface averaged 7 microm. A list of key parameters to ensure the comparability of surveys aiming at observing temporal or spatial deposition variation is presented.

Air Pollutants↗

Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

Cells, Cultured↗

Elemental content of lichens of the Point Reyes Peninsula, northern California.

The Point Reyes peninsula in northern California is about 45 km northwest of San Francisco and occasionally receives air masses from the city contributing to haze and lowered visibility. Although gaseous pollutants are not a problem, fine particulates containing carbon and other elements are measurable at the park. In this study, five lichen species were sampled along a 40-km linear transect from southeast to northwest from the town of Bolinas at the southern end of the peninsula. Elevated levels of Na and S and the Na/S ratio at localities nearer the ocean confirmed there is a definite marine aerosol influence on lichen elemental content. Elevated potassium and mercury concentrations were also found at high elevation sites that intercept fog. However, arsenic and lead were found to decrease with distance from Bolinas along the transect, suggesting these two elements originate in the fine particulate haze that enters the park from San Francisco. Concentrations of elements were not at enriched levels for other lichen species. A possible local influence of roads on element content needs further study.

Air Pollutants↗

Activation of p38 and N-acetylcysteine-sensitive c-Jun NH2-terminal kinase signaling cascades is required for induction of apoptosis in Parkinson's disease cybrids.

Cytoplasmic hybrid cells (cybrids) are created by selective amplification of mitochondrial genes against constant nuclear genetic and environmental backgrounds. Cybrids from patients with sporadic Parkinson's disease (PD) recapitulate disease features such as decreased complex I activity, increased oxidative stress, elevated activation of NF-kappaB, and production of Lewy body inclusions. We examined the activation of signaling pathways and NF-kappaB in PD cybrids after exposure to MAPK inhibitors and/or the antioxidant N-acetylcysteine (NAC). Under basal replicating conditions, PD cybrids have decreased viability that is associated with increased DNA condensation and poly-ADP ribose polymerase (PARP) cleavage as well as elevated p38 and JNK activity. Pharmacological inhibition of oxidative stress diminished the elevated p38, JNK activity and PARP cleavage, and enhanced PD cybrid viability. PD mitochondrial genes expressed in cybrids stimulate pro-apoptotic cell signaling and biochemistry through oxidative stress. These results support development of antioxidative therapeutics for PD.

Acetylcysteine↗

Altered intracellular signaling and reduced viability of Alzheimer's disease neuronal cybrids is reproduced by beta-amyloid peptide acting through receptor for advanced glycation end products (RAGE).

Activation of apoptosis by increased production of amyloid beta peptides (Abeta) has been implicated in neuronal cell death of Alzheimer's disease (AD). We used mitochondrial transgenic cybrid models of sporadic AD (SAD), which overproduce Abeta compared to control (CTL) cybrids, to investigate the effects of endogenously generated Abeta on intracellular signaling pathways and viability. Reducing SAD Abeta production with gamma-secretase inhibition altered the total phosphorylation profile of SAD cybrid to one similar to CTL cybrids and enhanced viability to approximately CTL cybrid levels. Treating CTL cybrids with exogenous Abeta or conditioned media (CM) from SAD cybrids activated the signaling pathways active in SAD cybrids under basal condition and decreased viability. Antibodies against receptor for advanced glycation end products (RAGE) blocked Abeta-induced activation of the p38, JNK pathways, and NF-kappaB in CTL cybrids and offered protection against the neurotoxic effects of Abeta. Expression of SAD mitochondrial genes in cybrids activates stress-related signaling pathways and reduces viability. This SAD phenotype is produced by endogenously generated Abeta and can be replicated by exogenous Abeta acting through RAGE.

Aged↗

Cyclical mitochondrial deltapsiM fluctuations linked to electron transport, F0F1 ATP-synthase and mitochondrial Na+/Ca+2 exchange are reduced in Alzheimer's disease cybrids.

Reduced complex IV, increased oxidative stress and beta amyloid peptide secretion in Alzheimer's disease (AD) can be replicated in cybrid models. We characterized cyclical mitochondrial deltapsiM fluctuations ('flickering') in neuroblastoma cells and AD/CTL cybrids. Flickering was blocked by ATP-synthase inhibition, was not observed in rho0 cells and was not blocked by antioxidant treatment. Flickering was not affected by the Ca(+2) uniporter antagonist Ru360 but was eliminated by BAPTA or CGP37137 blockade of the mitochondrial Na(+)/Ca(+2) exchanger. AD cybrid mitochondria showed reduced flickering. Flickering seems to represent coupling of deltapsiM to F0F1 ATP-synthase; reduction of flickering in AD cybrids suggests dysfunction of this coupling.

Alzheimer Disease↗

Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death.

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling.

Aged↗

Postmenopausal estrogen use affects risk for Parkinson disease.

BACKGROUND: Although estrogen therapy has been associated with improved cognitive functioning, a reduced risk of dementia in women with Parkinson disease (PD), and a decreased risk of Alzheimer disease, estrogen therapy has not affected the risk of PD per se. OBJECTIVE: To determine whether postmenopausal women with PD differed from control subjects with regard to estrogen exposure.Design, Setting, and Patients A case-control design was used, abstracting questionnaire data obtained via interview from 133 female PD cases and 128 female controls during routine outpatient clinic visits in 1999 at a mid-Atlantic tertiary care referral center. There were 140 subjects (68 PD cases and 72 controls) who met the inclusion criteria. Main Outcome Measure Use of postmenopausal estrogen therapy. RESULTS: More women in the control group than in the PD group took postmenopausal estrogen (36 [50%] of 72 women vs 17 [25%] of 68 women; P<.003), and women who had taken postmenopausal estrogen were less likely to develop PD than those who had not (odds ratio, 0.40 [95% confidence interval, 0.19-0.84]; P<.02). Among PD cases only, postmenopausal estrogen use was not associated with age of onset. CONCLUSION: Postmenopausal estrogen therapy may be associated with a reduced risk of PD in women.

Adult↗

Mitochondrial abnormalities in cybrid cell models of sporadic Alzheimer's disease worsen with passage in culture.

We created and studied new cybrid cell lines from sporadic Alzheimer's disease (SAD) or control (CTL) subjects to assess mitochondrial abnormalities just after metabolic selection ("early passage") and again six passages later ("late passage"). Cytochrome oxidase (CO) activities in early passage SAD cybrids created independently from the same platelet samples were highly correlated. Early passage SAD and CTL cybrids showed equivalent mitochondrial morphologies. Late passage SAD cybrids showed increased mitochondrial number, reduced mitochondrial size, and an approximately eightfold increase in morphologically abnormal mitochondria. Deficiency of SAD cybrid mitochondrial membrane potentials (DeltaPsi(M)) increased with passage. Mitochondrial bromodeoxyuridine (BrdU) uptake to estimate mitochondrial DNA (mtDNA) synthesis did not change with passage in CTL but increased in SAD cybrids. With time in culture, SAD mtDNA appears to replicate faster in cybrids, yielding cells with relative worsening of bioenergetic function. Metabolically deleterious SAD mitochondrial genes, like those in yeast, may have a replicative advantage over nondeleterious mitochondrial genes that assume dominance in CTL cybrids.

Aged↗

Development of mitochondrial gene replacement therapy.

Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.

Animals↗

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies.

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Aged↗

Elemental chemistry of four lichen species from the Apostle Islands, Wisconsin, 1987, 1995 and 2001.

Four lichen species sampled three times over a 15-year time span at four of the Apostle Islands, Wisconsin were analyzed for 16 chemical elements in order to determine time trends and spatial patterns. Factor analyses of the data revealed that elements associated with soils (Al, Cr, Fe, Na, Ni and S) have increased over the study period, while nutrient and pollutant elements (Cu, K, P, Pb and Zn) have decreased. Four other elements (Ca, Cd, Mg and Mn) were unchanged over time. Cladina rangiferina, a terricolous species, contained the lowest concentrations of all elements, while the corticolous species Evernia mesomorpha was highest in soil elements, Hypogymnia physodes was highest in Ca, Cd, Mg and Mn, and Parmelia sulcata was highest in the nutritional elements. Lichens on islands within 3-4 km of the mainland were highest in soil elements, which decreased with distance from the mainland. Elements that were 18-43% greater on the nearest islands were significantly different between near and far islands. Eight elements (Al, Ca, Cd, K, Mg, Mn, P and S) exceeded enrichment levels for Hypogymnia physodes, suggesting possible contamination problems for this species. Although Pb has decreased significantly over the time period, other elements have increased in the lichens while decreasing in the atmosphere, suggesting that accumulation in the environment is continuing.

Air Pollutants↗

Interactions among nitric oxide and Bcl-family proteins after MPP+ exposure of SH-SY5Y neural cells I: MPP+ increases mitochondrial NO and Bax protein.

We studied effects of methylpyridinium ion (MPP(+)) on apoptosis, cell death and regulation of Bcl-2-family proteins in SH-SY5Y neuroblastoma cells. MPP(+) increased intracellular accumulation of DNA-histone complexes as a measure of apoptosis and decreased intracellular calcein fluorescence as a measure of cell death. If ATP synthesis was supported, MPP(+) caused apoptosis in rho(0) cells devoid of electron transport function. Caspase inhibition blocked apoptosis but not cell death caused by MPP(+). MPP(+) increased levels of Bax, Bcl-2 and Bcl-X(L) proteins approximately 2-fold over 24 hr, with Bax increases occurring first; Bax did not increase in rho(0) cells. The Bax increase, but not that of Bcl-2 or Bcl-X(L), was dependent on nitric oxide (NO) and seemed post-transcriptional. DAF-FM imaging revealed increased mitochondrial NO within hours of exposure to MPP(+). Western blots showed a constitutive approximately 130 kD protein that stained for NOS-2, consistent with reports of mitochondrial nitric oxide synthase (mtNOS). MPP(+) caused a NO-dependent release of cytochrome C into cytoplasm. MPP(+) increases mitochondrial NO levels and causes a NO-dependent increase in Bax protein, providing a mechanism for NOS-and Bax-dependency of MPTP neurotoxicity in vivo and implicating locally produced NO as a signaling molecule used by mitochondria to manipulate cell death cascades.

Apoptosis↗

Interactions among nitric oxide and Bcl-family proteins after MPP+ exposure of SH-SY5Y neural cells II: exogenous NO replicates MPP+ actions.

In the preceding companion article, we showed that the neurotoxin methylpyridinium (MPP(+)) increases mitochondrial nitric oxide (NO), causes a post-transcriptional, NO-dependent increase in Bax protein and produces caspase-dependent apoptosis and caspase-independent cell death. In the present study, we show that exogenous NO replicates these findings. The long-term NO generator diethylenetriamine-NO (DETA-NO) reproduced the post-transcriptional Bax protein increase, but did not increase Bcl-2 or Bcl-X(L) proteins. Like MPP(+), DETA-NO caused an early decrease in Bcl-2 mRNA, did not increase Bax protein in rho(0) cells and caused caspase- and cycloheximide-dependent apoptosis and caspase-independent cell death. We developed cell lines with inducible overexpression of Bcl proteins, at levels relevant to those we found in cells exposed to MPP(+) or DETA-NO. Inducible overexpression ( approximately 2-fold) of Bcl-2 or Bcl-X(L) proteins reduced MPP(+) or NO-induced apoptosis but did not affect cell death. Inducible Bax overexpression ( approximately 5-fold) slightly increased cell death. Our results show that exogenous NO mimics actions of MPP(+) on SH-SY5Y neuroblastoma cells and supports the mediation of MPP(+) neurotoxicity by NO generated intracellularly in mitochondria.

Apoptosis↗

Dependence on electron transport chain function and intracellular signaling of genomic responses in SH-SY5Y cells to the mitochondrial neurotoxin MPP(+).

SH-SY5Y neuroblastoma cells exposed to the complex I inhibitor/parkinsonian neurotoxin methylpyridinium ion (MPP(+)) activate both survival and death-promoting signaling pathways and undergo MEK/ERK-dependent, phosphatidylinositol-3 kinase-dependent, and c-Jun kinase-dependent cell death. Because genomic responses to MPP(+) are not extensively characterized, we used nylon cDNA arrays to measure gene expression following exposure to an apoptosis-producing [MPP(+)]. Many changes occurred within 5 min, and all gene expression changes appeared before biochemical and morphological markers of apoptosis. The majority of gene expression changes in SY5Y were not found in rho(0) cells, indicating dependence of these changes on intact electron transport activity. rho(0) cells exposed to MPP(+) produced different expression profiles, indicating the potential for responses independent of complex I inhibition. MPP(+)-induced gene expression patterns in normal SY5Y cells were sensitive to inhibitors of MEK/ERK (UO 126) or phosphatidylinositol-3 kinase (LY 294002), demonstrating regulation of gene expression by these survival-promoting signaling pathways. The primary signaling molecules mediating these MPP(+)-induced gene expression changes are unknown but ultimately utilize MEK/ERK and phosphatidylinositol-3 kinase signaling. Genes suppressed by UO 126 or LY 294002 during MPP(+) exposure may mediate cell survival; those expressed in the presence of UO 126 or LY 294002 may mediate cell death in this in vitro model of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗

Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore.

Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester (TMRM). Calcein/TMRM-visualized mitochondrial depolarization was prevented by cyclosporin A (CsA) or approximately two-fold increased levels of BclXL protein. Dynamic imaging of mitochondrial potential (Deltapsi M) with TMRM showed that DETA-NO induced cycles of mitochondrial depolarization/repolarization ("flickering"). Fifteen-30 min of DETA-NO exposure caused high-frequency flickering with small peak size; 2 h of DETA-NO produced large peaks with prolonged depolarization. NO-induced flickering but not that from Bax was blocked by the calcium uniporter antagonist Ru360. Our findings show rapid-onset, dynamic regulation of Deltapsi M by NO, implying that neuroprotective therapies for brain ischemia target cell death processes downstream of effects of NO on mitochondria.

Animals↗

Reduction of oxidative stress in amyotrophic lateral sclerosis following pramipexole treatment.

Oxidative abnormalities have been identified both in familial amyotrophic lateral sclerosis (FALS) and the more prevalent sporadic ALS (SALS). Mitochondria dysfunction and toxic free radicals may play a role in this disease process, although the exact pathogenesis of both forms of ALS remains unknown. 2,3-DHBA is a hydroxylated salicylate by product that has been shown to be a reliable marker of increased free radical activity and is reliably assayed by HPLC. Following an oral salicylate load, we found elevated serum levels of 2, 3-dihydroxybenzoic acid (2,3-DHBA) and DHBA/salicylate in SALS subjects. Pramipexole has been shown to reduce oxidative stress and be neuroprotective in cell and animal models of neurodegeneration. We studied 12 SALS patients to determine the levels of 2,3-DHBA both before and after treatment with pramipexole. We found that pramipexole treatment up to 6 mg/day was well tolerated. The mean 2,3-DHBA serum levels were reduced by 45% and DHBA/salicylate ratios declined by 59% following treatment with pramipexole. SALS patients show apparent increases in systemic oxygen radical production that are reduced by pramipexole treatment at conventional doses, suggesting that pramipexole or related compounds may interrupt free radical production in SALS.

Aged↗

Stability of gene expression in postmortem brain revealed by cDNA gene array analysis.

We utilized nylon arrays to measure gene expression in mouse brains after various postmortem intervals (PMIs). Gene expression after overnight refrigeration or 4 h at room temperature then overnight refrigeration correlated highly and approximately equivalently to that in brains processed immediately. After 8-24 h at room temperature and overnight refrigeration gene expression correlation and equivalency declined, but 90-95% of detected genes were within +/-40% of baseline levels. Brain homogenate pH did not change with PMI.

Animals↗