Do naps benefit novel word learning? Developmental differences and white matter correlates.

Memory representations of newly learned words undergo changes during nocturnal sleep, as evidenced by improvements in explicit recall and lexical integration (i.e., after sleep, novel words compete with existing words during online word recognition). Some studies have revealed larger sleep-benefits in children relative to adults. However, whether daytime naps play a similar facilitatory role is unclear. We investigated the effect of a daytime nap (relative to wake) on explicit memory (recall/recognition) and lexical integration (lexical competition) of newly learned novel words in young adults and children aged 10-12 years, also exploring white matter correlates of the pre- and post-nap effects of word learning in the child group with diffusion weighted MRI. In both age groups, a nap maintained explicit memory of novel words and wake led to forgetting. However, there was an age group interaction when comparing change in recall over the nap: children showed a slight improvement whereas adults showed a slight decline. There was no evidence of lexical integration at any point. Although children spent proportionally more time in slow-wave sleep (SWS) than adults, neither SWS nor spindle parameters correlated with over-nap changes in word learning. For children, increased fractional anisotropy (FA) in the uncinate fasciculus and arcuate fasciculus were associated with the recognition of novel words immediately after learning, and FA in the right arcuate fasciculus was further associated with changes in recall of novel words over a nap, supporting the importance of these tracts in the word learning and consolidation process. These findings point to a protective role of naps in word learning (at least under the present conditions), and emphasize the need to better understand both the active and passive roles that sleep plays in supporting vocabulary consolidation over development.

Growing up with interfering neighbours: the influence of time of learning and vocabulary knowledge on written word learning in children.

Evidence suggests that new vocabulary undergoes a period of strengthening and integration offline, particularly during sleep. Practical questions remain, however, including whether learning closer to bedtime can optimize consolidation, and whether such an effect varies with vocabulary ability. To examine this, children aged 8-12-years-old (n 59) were trained on written novel forms (e.g. BANARA) in either the morning (long delay) or the evening (short delay). Immediately after training and the next day, lexical competition (a marker of integration) was assessed via speeded semantic decisions to neighbouring existing words (e.g. BANANA); explicit memory was measured via recognition and recall tasks. There were no main effects indicating performance changes across sleep for any task, counter to studies of spoken word learning. However, a significant interaction was found, such that children with poorer vocabulary showed stronger lexical competition on the day after learning if there was a short delay between learning and sleep. Furthermore, while poorer vocabulary was associated with slower novel word recognition speed before and after sleep for the long delay group, this association was only present before sleep for the short delay group. Thus, weak vocabulary knowledge compromises novel word acquisition, and when there is a longer period of post-learning wake, this disadvantage remains after a consolidation opportunity. However, when sleep occurs soon after learning, consolidation processes can compensate for weaker encoding and permit lexical integration. These data provide preliminary suggestion that children with poorer vocabulary may benefit from learning new words closer to bedtime.

Learning to live with interfering neighbours: the influence of time of learning and level of encoding on word learning.

New vocabulary is consolidated offline, particularly during sleep; however, the parameters that influence consolidation remain unclear. Two experiments investigated effects of exposure level and delay between learning and sleep on adults' consolidation of novel competitors (e.g. BANARA) to existing words (e.g. BANANA). Participants made speeded semantic decisions (i.e. a forced choice: natural versus man-made) to the existing words, with the expectation that novel word learning would inhibit responses due to lexical competition. This competition was observed, particularly when assessed after sleep, for both standard and high exposure levels (10 and 20 exposures per word; Experiment 1). Using a lower exposure level (five exposures; Experiment 2), no post-sleep enhancement of competition was observed, despite evidence of consolidation when explicit knowledge of novel word memory was tested. Thus, when encoding is relatively weak, consolidation-related lexical integration is particularly compromised. There was no evidence that going to bed soon after learning is advantageous for overnight consolidation; however, there was some preliminary suggestion that longer gaps between learning and bed-onset were associated with better explicit memory of novel words one week later, but only at higher levels of exposure. These findings suggest that while lexical integration can occur overnight, weaker lexical traces may not be able to access overnight integration processes in the sleeping brain. Furthermore, the finding that longer-term explicit memory of stronger (but not weaker) traces benefit from periods of wake following learning deserves examination in future research.

Preoperative breast MRI and mortality in older women with breast cancer.

PURPOSE: The survival benefit from detecting additional breast cancers by preoperative magnetic resonance imaging (MRI) continues to be controversial. METHODS: We followed a cohort of 4454 women diagnosed with non-metastatic breast cancer (stage I-III) from 2/2005-6/2010 in five registries of the breast cancer surveillance consortium (BCSC). BCSC clinical and registry data were linked to Medicare claims and enrollment data. We estimated the cumulative probability of breast cancer-specific and all-cause mortality. We tested the association of preoperative MRI with all-cause mortality using a Cox proportional hazards model. RESULTS: 917 (20.6%) women underwent preoperative MRI. No significant difference in the cumulative probability of breast cancer-specific mortality was found. We observed no significant difference in the hazard of all-cause mortality during the follow-up period after adjusting for sociodemographic and clinical factors among women with MRI (HR 0.90; 95% CI 0.72-1.12) compared to those without MRI. CONCLUSION: Our findings of no breast cancer-specific or all-cause mortality benefit supplement prior results that indicate a lack of improvement in surgical outcomes associated with use of preoperative MRI. In combination with other reports, the results of this analysis highlight the importance of exploring the benefit of preoperative MRI in patient-reported outcomes such as women's decision quality and confidence levels with decisions involving treatment choices.

Consolidating new words from repetitive versus multiple stories: Prior knowledge matters.

Prior knowledge is proposed to support the consolidation of newly acquired material. The current study examined whether children with superior vocabulary knowledge show enhanced overnight consolidation, particularly when new words are encountered in varying stories. Children aged 10 and 11 years (N = 42) were exposed to two sets of eight spoken novel words (e.g., "crocodol"), with one set embedded in the same story presented twice and the other presented in two different stories. Children with superior vocabulary knowledge showed larger overnight gains in explicit phonological and semantic knowledge when novel words had been encountered in multiple stories. However, when novel words had been encountered in repetitive stories, existing knowledge exerted no influence on the consolidation of explicit phonological knowledge and had a negative impact on the consolidation of semantic knowledge. One full day (24 h) after story exposure, only very weak evidence of lexical integration (i.e., slower animacy decisions toward the basewords [e.g., "crocodile"] than toward the control words) was observed for novel words learned via repetitive (but not multiple) stories. These data suggest that although the consolidation of explicit new word knowledge learned through multiple contexts is supported by prior knowledge, lexical integration might benefit more from repetition.

Eye-tracking the time-course of novel word learning and lexical competition in adults and children.

Lexical competition is a hallmark of proficient, automatic word recognition. Previous research suggests that there is a delay before a new spoken word becomes engaged in this process, with sleep playing an important role. However, data from one method - the visual world paradigm - consistently show competition without a delay. We trained 42 adults and 40 children (aged 7-8) on novel word-object pairings, and employed this paradigm to measure the time-course of lexical competition. Fixations to novel objects upon hearing existing words (e.g., looks to the novel object biscal upon hearing "click on the biscuit") were compared to fixations on untrained objects. Novel word-object pairings learned immediately before testing and those learned the previous day exhibited significant competition effects, with stronger competition for the previous day pairings for children but not adults. Crucially, this competition effect was significantly smaller for novel than existing competitors (e.g., looks to candy upon hearing "click on the candle"), suggesting that novel items may not compete for recognition like fully-fledged lexical items, even after 24h. Explicit memory (cued recall) was superior for words learned the day before testing, particularly for children; this effect (but not the lexical competition effects) correlated with sleep-spindle density. Together, the results suggest that different aspects of new word learning follow different time courses: visual world competition effects can emerge swiftly, but are qualitatively different from those observed with established words, and are less reliant upon sleep. Furthermore, the findings fit with the view that word learning earlier in development is boosted by sleep to a greater degree.

Accessing and selecting word meaning in autism spectrum disorder.

BACKGROUND: Comprehension difficulties are commonly reported in autism spectrum disorder (ASD) but the causes of these difficulties are poorly understood. This study investigates how children with ASD access and select meanings of ambiguous words to test four hypotheses regarding the nature of their comprehension difficulties: semantic deficit, weak central coherence, reduced top-down control and inhibition deficit. METHODS: The cross-modal semantic priming paradigm was used. Children heard homonym primes in isolation or as final words in sentences biased towards the subordinate meaning and then named picture targets depicting dominant or subordinate associates of homonyms. RESULTS: When homonyms were presented in isolation, children with ASD and controls showed priming for dominant and subordinate pictures at 250ms ISI. At 1,000ms ISI, the controls showed dominant (but not subordinate) priming whilst the ASD group did not show any priming. When homonyms were presented in subordinate sentence contexts, both groups only showed priming for context-appropriate (subordinate) meanings at 250ms ISI, suggesting that context has an early influence on meaning selection. At 1,000ms ISI the controls showed context-appropriate (but not inappropriate) priming whereas the ASD group showed both appropriate and inappropriate priming. CONCLUSIONS: Children with ASD showed intact access to semantic information early in the time course of processing; however, they showed impairments in the selection of semantic representations later in processing. These findings suggest that a difficulty with initiating top-down strategies to modulate online semantic processing may compromise language comprehension in ASD. Implications for intervention are discussed.

Adrenal lipid profiles of chemically sympathectomized normoxic and hypoxic neonatal rats.

Neonatal hypoxia is a common condition that elicits a coordinated endocrine response. In the neonatal rat, hypoxia induces an ACTH-independent increase in corticosterone which can be partially blocked by chemical sympathectomy. The present study sought to characterize the effects of sympathectomy on the adrenal lipid profile, since previous work suggested that augmented plasma corticosterone during hypoxia may be due to changes in adrenal lipid metabolism. Newborn rats were exposed to normoxia or hypoxia from birth to seven days of age, and guanethidine was used to produce the sympathectomy. Plasma epinephrine and norepinephrine were not significantly affected by hypoxia, while guanethidine decreased plasma norepinephrine in normoxic and hypoxic pups. Hypoxia alone increased the concentration of cholesterol esters in the adrenal gland; this increase was due to increases in cholesterol ester-associated oleic (18:1n9), docosahexaenoic (22:6n3), arachidonic (20:4n6), and adrenic (22:4n6) acids. Hypoxia also increased diglyceride-associated adrenic acid. Guanethidine treatment attenuated the hypoxia-induced increase in cholesterol ester-bound arachidonic and adrenic acids. Guanethidine also decreased saturated fatty acid concentrations and increased n3 fatty acid-enriched triglycerides. The results support the idea that the ACTH-independent corticosterone response to hypoxia in the neonatal rat is mediated by specific, sympathetically driven alterations in the adrenal lipid profile.

Proton conduction through full-length gp91phox requires histidine 115.

The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 (phox), one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 (phox) has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 (phox) has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 (phox). Stable CHO cell lines were established which expressed full-length gp91 (phox) in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 (phox) and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 (phox). The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 (phox). Therefore, histidine 115 is required for proton conduction by both full-length gp91 (phox) and the N-terminal 230-amino-acid fragment of gp91 (phox). Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115.

NADPH oxidase subunit gp91phox: a proton pathway.

The generation of superoxide by the NADPH oxidase is an electrogenic process resulting in a rapid depolarisation of the membrane potential of the cell. The efflux of H+ ions through an arachidonate-activatable, Zn(2+)-inhibitable H+ pathway accompanies the efflux of electrons and provides the necessary charge compensation. Inhibition of H+ flux leads to inhibition of superoxide generation. The protein gp91phox, a transmembrane component of the NADPH oxidase, was demonstrated to be capable of acting as the NADPH oxidase-associated H+ channel in a stable CHO cell line, CHO91. The N-terminal 230 amino acids contain all that is required for the protein to form an H+ channel and specifically histidine 115 is important to the ability of gp91phox to conduct H+ ions. The recording of outward currents from CHO91 cells, in the whole-cell configuration, demonstrated that gp91phox is also capable of functioning as a voltage-gated H+ conductance pathway. The similarity in properties between voltage-elicited outward currents, from both wild type and the mutations, and the arachidonate-activated H+ flux strongly suggests that these H+ pathways are one in the same. Among the recently identified homologues of gp91phox only NOH-1S has so far been demonstrated to also act as an H+ conductance pathway.

New technology for improved vaccine safety and efficacy.

Nearly all of the 2000 vaccines presently licensed by the US Department of Agriculture for veterinary use in the United States are conventional vaccines containing either killed or modified live whole bacteria or viruses. Recent advances in molecular biology, immunology, microbiology, and genetics and in understanding microbial pathogenesis have led to the development of a wide variety of new approaches for developing safer and more effective vaccines. This article briefly describes these new technologies and their potential advantages and disadvantages as compared with conventional killed and modified live vaccines.

Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid.

1. Effects of arachidonic acid (AA) on proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, using an applied NH4+ gradient to control pH(i). 2. Superoxide anion (O2-) release was assessed by cytochrome c reduction in human eosinophils. Significant O2- release was stimulated by 5-10 microM AA. 3. AA activated diphenylene iodinium (DPI)-inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (I(e)) were elicited in human eosinophils at AA concentrations (3-10 microM) similar to those that induced O2- release. 4. The voltage-gated proton conductance (g(H)) in eosinophils stimulated with AA was profoundly enhanced: H+ current amplitude (I(H)) increased 4.6 times, activation was 4 times faster, and the H+ conductance-voltage (g(H)-V) relationship was shifted to substantially more negative voltages. The electrophysiological effects of AA resembled those reported for PMA, except that AA did not consistently slow tau(tail) (deactivation of H+ currents). 5. The stimulation of both proton and electron currents by AA was reversible upon washout. Repeated exposure elicited repeated responses. The activation of H+ currents by AA was dissociable from its activation of NADPH oxidase; H+ currents were enhanced at low concentrations of AA that did not elicit detectable I(e) or when NADPH oxidase was inhibited by DPI. 6. Most of the effects of AA on H+ currents qualitatively resemble those reported in whole-cell studies, reflecting a more direct action than PMA. The results are compatible with AA being an immediate activator of both NADPH oxidase and proton channels in human eosinophils.

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites.

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

Identifying and characterizing one-time and never users of mammography screening services among medicare eligible women.

BACKGROUND: Despite eligibility for a screening mammogram once every 2 years from 1991 to 1997, only a small percentage of Medicare women utilized this benefit. We examined mammography use among 388,707 North Carolina Medicare women from 1994 to 1997 to identify characteristics of one-time and never users of mammography. METHODS: Data were obtained from North Carolina Medicare mammography claims and enrollment files from 1994 to 1997. Women ages 65+ as of 01/01/1994, continuously enrolled in Medicare from 1994 to 1997, and alive as of 12/31/1997 were included in the sample (n = 388,707). Mammogram use was categorized as never, once, or at least twice during 1994/1995 and 1996/1997. Women with at least one mammography claim during 1994/1995 and at least one mammography claim during 1996/1997 were called repeat users, women with one mammography claim during the 4 years were labeled one-time users, and women with zero mammography claims during the 4 years were termed never users. Multivariate logistic regression analyses were conducted to determine associations between characteristics and mammography frequency. RESULTS: Biennial mammography claims data rates were 35.3% in 1994/1995 and 41.8% in 1996/1997. Compared with all other users, one-time users (n = 108,899) were more likely to be ages 65-74 (vs 75-84 and 85+), live in an urban versus rural county, and be eligible for Medicare only versus Medicare and Medicaid. Never users (n = 184,545) were more likely to be ages 85+, be non-Caucasian, live in a rural county, and be eligible for both Medicare and Medicaid versus Medicare. CONCLUSIONS: These results demonstrate different demographic characteristics for one-time and never mammography users. This approach of using multiple years of claims data to segment the targeted population provides the opportunity to tailor interventions to subgroups.

Surface electrode somatosensory-evoked potentials in spinal surgery: implications for indications and practice.

STUDY DESIGN: A retrospective study of 442 major spinal operations with spinal cord monitoring performed in a University Hospital between 1982 and 1992 was performed. OBJECTIVES: To assess the reliability of the authors' method for monitoring by somatosensory-evoked potential recording, to determine criteria for intraoperative corrective action, and to redefine the need for the wake-up test. SUMMARY OF BACKGROUND DATA: The routine use of somatosensory-evoked potential monitoring in spinal surgery remains controversial. In Nottingham, the authors have used a method of recording from either scalp or high cervical electrodes. METHODS: The recordings and outcomes of all monitored spinal operations between the years 1982 and 1992 were reviewed. RESULTS: In 442 procedures, 23 technical failures (no reliable monitoring) occurred. Most technical failures were in patients with severe preoperative neurology, identifiable by somatosensory-evoked potential recording before operation. In the remaining 419 procedures, a significant intraoperative change in response occurred in 70 procedures (16.7%). Using the definitions of the American EEG Society, the authors identified 10 true-positives and 60 false-positives. There were no false-negatives. A wake-up test was performed if an amplitude drop greater than 50% from baseline value persisted after attempts to correct any possible identifiable intraoperative cause. This occurred in only 21 patients (5%). In the true-positive group, somatosensory-evoked potential recordings remained persistently abnormal despite an apparently normal subsequent wake-up test. The sensitivity of the method according to current definitions was 100% and the specificity 85.33%. CONCLUSIONS: Modified guidelines are needed for routine intraoperative use of somatosensory-evoked potential monitoring in spinal surgery. Such guidelines should avoid the term "false-positive" as currently defined and concentrate on the causative analysis of abnormal responses that warn of critical spinal cord dysfunction before that becomes irreversible and allow for appropriate action. Information from this monitoring method alerted the surgeon to the possible need for corrective action in an additional 9.78% of the reported patients, who traditionally would have been regarded as false-positives. A wake-up test still is indicated in patients with persistent suppression of their somatosensory-evoked potential despite correction of any identifiable cause and in cases of technical failure. The current method proved flexible, versatile, and reliable for future use.

Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group.

Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity.

Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway.

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.

Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel.

The efflux of protons through a H+ channel acts as the charge compensation pathway for the electrogenic generation of superoxide (O-2) by human neutrophil NADPH oxidase. It has previously been shown that the N-terminal 230 amino acids of the product of the X-linked chronic granulomatous gene gp91(phox) contain all that is required for it to function as the arachidonate-activable, NADPH oxidase-associated H+ channel (Henderson, L. M., Thomas, S., Banting, G., and Chappell, J. B. (1997) Biochem. J. 325, 701-705). To identify functionally important amino acids, Chinese hamster ovary (CHO) cell lines were constructed that expressed point mutations in the N terminus of gp91(phox). No H+ flux was observed in CHO cell lines expressing the N-terminal gp91(phox) mutants H111L, H115L, and H119L, or H115L, or H115K. Partial retention of H+ channel function was, however, observed in the H115D CHO cell line. The addition of arachidonic acid to R91E,R92E CHO cells elicited a full H+ channel response. The buffering capacity and response of 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to H+ were the same in all cell lines. Therefore, it can be concluded that His-115 is important to the ability of gp91(phox) to function as the NADPH oxidase-associated H+ channel and that the mechanism of H+ conduction involves protonation and deprotonation of an amino acid with an appropriate pK value.