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J W Allen

Publications and source records attributed to J W Allen.

At least 55 records · Page 3Linked to original sources
PubMed2000

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior.

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.

Allosteric Regulation↗
PubMed2000

Foreign metallothionein-I expression by transient transfection in MT-I and MT-II null astrocytes confers increased protection against acute methylmercury cytotoxicity.

The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular Na(2)51CrO(4) efflux and D-[2,3-3H]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 microM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p<0.01) increase in Na(2)51CrO(4) efflux and a significant (p<0.05) decrease in the initial rate (1 min) of D-[2, 3-3H]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly (p<0.01) decreased the effect of MeHg on Na(2)51CrO(4) efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 microM) were indistinguishable. These results demonstrate that: (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg.

Animals↗
PubMed2000

Economic evaluation of use of diphtheria, tetanus, and acellular pertussis vaccine or diphtheria, tetanus, and whole-cell pertussis vaccine in the United States, 1997.

OBJECTIVE: To compare the economic costs and benefits associated with using either diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP) or diphtheria and tetanus toxoids and whole-cell pertussis vaccine (DTwP) in the United States in 1997. DESIGN: Standard cost-benefit analysis, from both the societal and health care system perspectives, was performed for each combination vaccine as well as for the pertussis components singly. SETTING: A simulated cohort of 4.1 million children from birth to age 15 years. MAIN OUTCOME MEASURES: Net costs (savings) and benefit-cost ratios (BCRs) RESULTS: Without a vaccination program, diphtheria, tetanus, and pertussis disease caused more than 3 million cases and more than 28,000 deaths, at a cost of $23.6 billion. From the societal perspective, net savings because of the use of DTaP and DTwP were $22.510 million and $22.623 million, respectively. The net savings from the acellular pertussis component and the whole-cell pertussis component only were $4.362 million and $4.474 million, respectively. Benefit-cost ratios for DTaP from a societal and health care system perspective were 27:1 and 9:1, respectively. Sensitivity analyses of key variables did not result in appreciable changes in results. CONCLUSIONS: Compared with no program, vaccination with DTaP or DTwP resulted in substantial savings, regardless of the perspective taken and for all sensitivity analyses conducted. Compared with DTwP, use of DTaP generated a small cost increase that might be offset by the value of other factors, such as increased confidence in pertussis vaccination resulting from reduced adverse events. Arch Pediatr Adolesc Med. 2000;154:797-803

Adolescent↗
PubMed2000

A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis.

Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates. Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle. Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis. A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with 'switched' axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine. This activated conformer returns over 20 min at 25 degrees C to the state previously observed for oxidized 'as isolated' cd1, which is catalytically inactive towards the same substrates.

Catalysis↗
PubMed2000

Activation of group I metabotropic glutamate receptors reduces neuronal apoptosis but increases necrotic cell death in vitro.

Glutamate released during acute CNS insults acts at metabotropic glutamate receptors (mGluR), including group I mGluR. Blockade of group I mGluR during in vitro neuronal trauma provides neuroprotection, whereas activation exacerbates such injury. However, the effects of group I mGluR agonists or antagonists have been primarily studied in in vitro models characterized by necrotic cell death. We examined the role of group I mGluR in the modulation of neuronal injury induced during oxygen-glucose deprivation (OGD), a well-studied model of necrosis, and by application of two well established pro-apoptotic agents: staurosporine and etoposide. Inhibition of group I mGluR attenuated necrosis induced by OGD, whereas selective activation of group I mGluR exacerbated such injury. In contrast, activation of group I mGluR, including selective activation of mGluR5, significantly attenuated apoptotic cell death induced by both staurosporine and etoposide. This effect was completely reversed by co-application of a group I mGluR antagonist. Thus, group I mGluR appear to exhibit opposite effects on necrotic and apoptotic neuronal cell death. Our findings suggest that activation of mGluR1 exacerbates neuronal necrosis whereas both mGluR1 and mGluR5 play a role in attenuation of neuronal apoptosis.

Animals↗
PubMed2000

Bilirubin oxidation by brain mitochondrial membranes is not affected by hyperosmolality.

Brain mitochondrial membranes oxidize bilirubin at a rate that may be biologically significant. Hyperosmolality delays clearance of bilirubin from rat brain. We hypothesized that this might be due to a decrease in brain bilirubin metabolism. Rats were anesthetized and infused with 75 mM/kg urea i.v. over 5 min (n = 7) and sacrificed at 1 h; control rats (n = 7) had no treatment. Crude mitochondrial membrane fractions ('P2') were produced by homogenization and differential centrifugation in sucrose. The change in optical density at 440 nM of a 10-micromol/l bilirubin solution was measured after 60 min incubation with mitochondrial membranes. Hyperosmolality did not affect bilirubin oxidation by brain mitochondrial membranes (t = 1.27, p = 0.23 by unpaired t-test). The lack of effect of hyperosmolality on bilirubin oxidation by brain mitochondrial membranes suggests that decreased clearance of bilirubin from brain in hyperosmolality may be related to changes in transfer of bilirubin across the blood-brain barrier.

Animals↗
PubMed2000

Astrocytes in methylmercury, ammonia, methionine sulfoximine and alcohol-induced neurotoxicity.

Astrocytes occupy approximately 25% of the CNS volume. Their "foot" processes are closely associated with synapses, nodes of Ranvier, axonal tracts, and capillaries. Astrocytic functions include neurotrophic factor secretion, control of extracellular pH, inactivation of glutamate, as well as uptake and metabolism of neurotransmitters. Astrocyte-neuron interactions provide strategic sites for actions of numerous chemical compounds. In this manuscript, we discuss examples of toxins that directly affect astrocyte function (methylmercury, ammonia, methionine sulfoximine, and alcohol), leading to an altered homeostatic control of the extracellular milieu and neuronal dysfunction. In addition, the potential role of astrocytic proteins, the metallothioneins, in attenuating the neurotoxicity of methylmercury is discussed.

Animals↗
PubMed1999

Transfection and overexpression of metallothionein-I in neonatal rat primary astrocyte cultures and in astrocytoma cells increases their resistance to methylmercury-induced cytotoxicity.

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.

Animals↗
PubMed1999

Factors determining normalization of pulmonary vascular resistance following successful balloon mitral valvotomy.

Balloon mitral valvotomy (BMV) provides improvement in pulmonary vascular resistance (PVR) in patients with severe mitral stenosis. Its normalization, however, remains questionable. We evaluated PVR before, after BMV, and at follow-up in 37 patients who had a previous successful BMV. Patients were divided into 2 groups: group 1 had 21 patients with normalized PVR (<125 dynes/s/cm5) either after BMV or at follow-up, and group 2 had 16 patients with persistently abnormal PVR. Patients in group 2 were older than patients in group 1 (55+/-13 vs 43+/-14 years, p = 0.01) and had atrial fibrillation more frequently (10 [63%] vs 6 [29%], p = 0.04). Age, cardiac rhythm, mitral valve area, pulmonary bed gradient, pulmonary artery pressure, and PVR before the procedure were significant univariate predictors for normalization of PVR. Age, echocardiographic score, systolic pulmonary artery pressure, and mitral regurgitation were all independent determinants of normalization of PVR in a multivariate logistic regression model. We conclude that PVR failed to return to normal in 16 patients (43%) after successful BMV; this can be predicted by baseline clinical and hemodynamic parameters.

Adult↗
PubMed1999

PEEP-induced pulmonary vasoconstriction in neonatal piglets: analysis by pressure-flow curves.

We analyzed the effect of two levels of positive end-expiratory pressure (PEEP: 10 and 15 cm H2O) on pulmonary hemodynamics in neonatal piglet lungs isolated in situ and perfused extracorporeally using pulmonary artery pressure-flow (Pa/Q) relationships. Pulmonary artery pressure (Pa) was measured at flow rates of 50, 75, 100, 125, and 150 mL/kg/min. Pa/Q relationship was evaluated by the slope of the Pa/Q plot and the zero-flow intercept pressure (Pi). Pa/Q relationship with PEEP was studied before and after verapamil. Both levels of PEEP increased the slope of the Pa/Q plot and Pi. PEEP of 15 cm H2O resulted in a steeper slope and a higher Pi compared to 10 cm H2O of PEEP (P < 0.05). Verapamil abolished the increase in slope of the pulmonary artery Pa/Q plot but did not affect the increase in Pi with PEEP. The increase in Pi was equal to the increase in mean airway pressure. Verapamil did not affect changes in ventilatory parameters. PEEP increased pulmonary vascular resistance (PVR) both by increasing the Pi, which reflects the weighted average of the critical closing pressure, and represents a "Starling resistor" phenomenon, and an increase in the slope of the P-Q plot, reflecting an increase in pulmonary vascular tone. This response may be unique to the neonatal pulmonary circulation.

Analysis of Variance↗
PubMed1999

Oxidation of bilirubin in the brain-further characterization of a potentially protective mechanism.

Bilirubin is a well-known neurotoxin and presents a particular problem in newborn infants. This is partly due to the high incidence of unconjugated hyperbilirubinemia in that age group, but may also be due to increased vulnerability to bilirubin toxicity. The brain may be able to protect itself against bilirubin toxicity through a process of oxidation. The responsible enzyme is localized on the inner mitochondrial membrane and appears to be more active in glia than in neurons and to increase in activity with postnatal maturation. Here we have investigated the possibility that the responsible enzyme might be a cytochrome oxidase, malate dehydrogenase, or monoamine oxidase, all enzymes located on the inner mitochondrial membrane. Mitochondria were obtained from rat brains through homogenization and differential centrifugation in sucrose medium. The ability of mitochondrial membranes to oxidize bilirubin was measured by following the change in optical density at 440 nm of a bilirubin solution to which a membrane suspension had been added. The activity was not changed by in vitro inhibitors of malate dehydrogenase or monoamine oxidase, but was moderately inhibited by ketoconazole and clotrimazole, both known inhibitors of hepatic cytochrome P450 oxidases. Activity was inhibited by depletion of cytochrome c in the mitochondria and reconstituted by reintroducing cytochrome c into the reaction mixture. The reaction was not modified by the addition of a free radical quencher, but was inhibited by removal of oxygen from the reaction mixture. The activity was significantly inhibited by cyanide. Activity was retained in a 100,000-g pellet and was not influenced by the addition of NAD, NADP, NADH, NADPH, GSH, or GSSH to this pellet. We conclude that the bilirubin-oxidizing activity in brain mitochondrial membranes is cytochrome c dependent, but does not appear to be unequivocally identifiable as a cytochrome P450 oxidase.

Animals↗
PubMed1999

Beta-amyloid-induced apoptosis of cerebellar granule cells and cortical neurons: exacerbation by selective inhibition of group I metabotropic glutamate receptors.

Administration of beta-amyloid fragment 25-35 (Abeta25-35) to cultured rat cerebellar granule cells (CGC) or cortical neurons caused cell death that was characterized by morphological and nuclear changes consistent with apoptosis. Inhibition of NMDA receptors produced a mild exacerbation of Abeta25-35 toxicity in cortical neurons; a similar effect was induced by AMPA/kainate receptor inhibition in CGC. Selective activation of group I metabotropic glutamate receptors (mGluR) by dihyroxyphenylglycine (DHPG) had no effect on Abeta25-35-induced apoptosis in either cell type, and was unaffected by blockade of ionotropic glutamate receptors. In contrast, selective inhibition of group I mGluR by (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) exacerbated Abeta toxicity in cortical neurons, whereas this treatment was without effect on CGC. However, AIDA significantly increased Abeta-induced apoptosis in CGC in the presence of either NMDA or AMPA/kainate receptor inhibition; blockade of both ionotropic glutamate receptor classes further increased the exacerbation of apoptosis following treatment with AIDA. These findings suggest that Abeta25-35-induced neuronal injury leads to activation of group I mGluR, which attenuates the resulting apoptosis.

Amyloid beta-Peptides↗
PubMed1999

Completion of meiosis is not always required for acrosome formation in HSP70-2 null mice.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase. However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Acrosome↗
PubMed1999

Combined mechanical trauma and metabolic impairment in vitro induces NMDA receptor-dependent neuronal cell death and caspase-3-dependent apoptosis.

Neuronal necrosis and apoptosis occur after traumatic brain injury (TBI) in animals and contribute to subsequent neurological deficits. In contrast, relatively little apoptosis is found after mechanical injury in vitro. Because in vivo trauma models and clinical head injury have associated cerebral ischemia and/or metabolic impairment, we transiently impaired cellular metabolism after mechanical trauma of neuronal-glial cultures by combining 3-nitropropionic acid treatment with concurrent glucose deprivation. This produced greater neuronal cell death than mechanical trauma alone. Such injury was attenuated by the NMDA receptor antagonist dizocilpine (MK801). In addition, this injury significantly increased the number of apoptotic cells over that accruing from mechanical injury alone. This apoptotic cell death was accompanied by DNA fragmentation, attenuated by cycloheximide, and associated with an increase in caspase-3-like but not caspase-1-like activity. Cell death was reduced by the pan-caspase inhibitor BAF or the caspase-3 selective inhibitor z-DEVD-fmk, whereas the caspase-1 selective inhibitor z-YVAD-fmk had no effect; z-DEVD-fmk also reduced the number of apoptotic cells after combined injury. Moreover, cotreatment with MK801 and BAF resulted in greater neuroprotection than either drug alone. Thus, in vitro trauma with concurrent metabolic inhibition parallels in vivo TBI, showing both NMDA-sensitive necrosis and caspase-3-dependent apoptosis.

Animals↗
PubMed1999

Metallothioneins attenuate methylmercury-induced neurotoxicity in cultured astrocytes and astrocytoma cells.

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by Northern blot and immunoprecipitation analyses, respectively. The ability of cells overexpressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na2(51)CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2.5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na2(51)CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MTs offer effective cellular adaptation of MeHg cytotoxicity.

Animals↗
PubMed1999

Glial cells in neurotoxicity development.

Neuroglial cells of the central nervous system include the astrocytes, oligodendrocytes, and microglia. Their counterparts in the peripheral nervous system are the Schwann cells. The term neuroglia comes from an erroneous concept originally coined by Virchow (1850), in which he envisioned the neurons to be embedded in a layer of connective tissue. The term, or its shortened form--glia, has persisted as the preferred generic term for these cells. A reciprocal relationship exists between neurons and glia, and this association is vital for mutual differentiation, development, and functioning of these cell types. Therefore, perturbations in glial cell function, as well as glial metabolism of chemicals to active intermediates, can lead to neuronal dysfunction. The purpose of this review is to explore neuroglial sites of neurotoxicant actions, discuss potential mechanisms of glial-induced or glial-mediated central nervous system and peripheral nervous system damage, and review the role of glial cells in neurotoxicity development.

Animals↗