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UNLABELLED: Student attendance in American public schools is a critical factor in securing limited operational funding. Student and teacher attendance influence academic performance. Limited data exist on indoor air and environmental quality (IEQ) in schools, and how IEQ affects attendance, health, or performance. This study explored the association of student absence with measures of indoor minus outdoor carbon dioxide concentration (dCO(2)). Absence and dCO(2) data were collected from 409 traditional and 25 portable classrooms from 22 schools located in six school districts in the states of Washington and Idaho. Study classrooms had individual heating, ventilation, and air conditioning (HVAC) systems, except two classrooms without mechanical ventilation. Classroom attributes, student attendance and school-level ethnicity, gender, and socioeconomic status (SES) were included in multivariate modeling. Forty-five percent of classrooms studied had short-term indoor CO(2) concentrations above 1000 p.p.m. A 1000 p.p.m. increase in dCO(2) was associated (P < 0.05) with a 0.5-0.9% decrease in annual average daily attendance (ADA), corresponding to a relative 10-20% increase in student absence. Annual ADA was 2% higher (P < 0.0001) in traditional than in portable classrooms. PRACTICAL IMPLICATIONS: This study provides motivation for larger school studies to investigate associations of student attendance, and occupant health and student performance, with longer term indoor minus outdoor CO(2) concentrations and more accurately measured ventilation rates. If our findings are confirmed, improving classroom ventilation should be considered a practical means of reducing student absence. Adequate or enhanced ventilation may be achieved, for example, with educational training programs for teachers and facilities staff on ventilation system operation and maintenance. Also, technological interventions such as improved automated control systems could provide continuous ventilation during occupied times, regardless of occupant thermal comfort demands.

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Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.

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PURPOSE: To determine the acute histologic and ultrastructural effects of a recently developed muscle-specific immunotoxin, ricin-mAb 35. METHODS: Graduated doses of ricin-mAb 35, composed of ricin conjugated to a monoclonal antibody against the nicotinic acetylcholine receptor of skeletal muscle, were injected into one superior rectus muscle in rabbits. After 3, 7, and 14 days, both superior rectus muscles were removed and prepared for electron microscopy and histologic examination, by using a number of immunohistochemical markers to identify inflammatory cell infiltration, muscle fiber loss, and muscle regeneration. RESULTS: Myotoxicity of the ricin-mAb 35 was focal and dose related. At the highest dose tested, there was substantial inflammatory cell infiltrate by 3 days, which largely disappeared by 7 days. Significant muscle loss was apparent by 7 days after ricin-mAb 35 treatment. Both the inflammatory reaction and muscle fiber loss were confined to the immediate injection site. Surrounding muscle appeared to be normal. At 14 days after treatment, early signs of muscle regeneration were evident within the tissue sections. No evidence of orbital or systemic toxicity was seen in any animal. CONCLUSIONS: Direct injection of ricin-mAb 35 into the extraocular muscles of rabbits results in a dose-related focal injury to the muscles, with a self-limited inflammatory component and significant muscle fiber loss. This novel immunotoxin may be useful in the treatment of strabismus if chronic studies show a sustained histologic and electrophysiologic effect.

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Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.

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Metropolis Monte Carlo simulations have been performed on four substructures from the cell-wall polysaccharide antigen of Streptococcus group A to explore the conformational behaviour of these compounds. The compounds examined are the trisaccharide, propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyranosyl)- alpha-L-rhamnopyranoside, 1, the tetrasaccharide, propyl 3-O-(3-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2-O-(alpha-L-rhamnopyranosyl)-alpha-L-rhamnopyranosyl)-alpha-L- rhamnopyranoside, 2, the hexasaccharide, propyl 3-O-(2-O-(3-O-(3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-L- rhamnopyranosyl)-alpha-L-rhamnopyranosyl)-3-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-alpha-L-rhamnopyranosyl)-alpha-L-rhamnopyranoside, 3, and the hexasaccharide, propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(3-O-(3-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyranosyl)-alpha-L- rhamnopyranosyl)-alpha-L-rhamnopyranosyl)-alpha-L-rhamnopyranoside, 4. In general, the conformational flexibility of similar glycosidic linkages in different compounds is comparable. However, in a few cases, small differences in the conformations available to these linkages in different structural environments could be detected. Interestingly, a second conformation found for the beta-D-GlcNAc-(1-->3)-alpha-L-Rha linkage in three of the compounds was not populated in the hexasaccharide 4. Furthermore, a conformational locale of the alpha-L-Rha-(1-->3)-alpha-L-Rha linkage found to be populated in the trisaccharide 1, tetrasaccharide 2, and hexasaccharide 4 is negligibly populated in the hexasaccharide 3. Ensemble averaged proton-proton distances compare favourably with experimental average distances obtained from NMR spectroscopy. The trisaccharide branch point in the hexasaccharides is shown to be a highly defined conformational feature. The same unit has been found to be one of the crucial elements recognized by anti-Group A Streptococcus antibodies, a result that has implications for the design of improved immunodiagnostics and vaccines.

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Two new radon mitigation techniques are introduced and their evaluation in a field study complemented by numerical model predictions is described. Based on numerical predictions, installation of a sub gravel membrane at the study site resulted in a factor of 2 reduction in indoor radon concentrations. Experimental data indicated that installation of "short-circuit" pipes extending between the subslab gravel and outdoors caused an additional factor of 2 decrease in the radon concentration. Consequently, the combination of these two passive radon mitigation features, called the membrane and short-circuit (MASC) technique, was associated with a factor of 4 reduction in indoor radon concentration. The energy-efficient active radon mitigation method, called efficient active subslab pressurization (EASP), required only 20% of the fan energy of conventional active subslab depressurization and reduced the indoor radon concentration by approximately a factor of 15, including the numerically-predicted impact of the sub-gravel membrane.

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Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was de-O-acylated, dephosphorylated, reduced, de-N-acylated, N-acetylated, and the products were separated by high-performance anion-exchange chromatography (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN-disaccharide lipid A backbone. Its structure was established by compositional and methylation analyses, and extensive NMR investigations including 1H,1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY), as well as heteronuclear 13C,1H COSY. In another reaction sequence the lipopolysaccharide was hydrolysed with dilute acetic acid and reduced with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3-deoxy-D-manno-octulosonic acid (Kdo) residue. A major product, 2, was isolated and investigated by the same methods as described for the decasaccharide 1. The following structures are proposed for compounds 1 and 2: alpha-D-GlcNAcp-(1-7)-[beta-D-Galp-(1-3)-]-alpha-Hepp-(1-2)- alpha-Hepp- (1-3)-[beta-D-Glcp-(1-4)-]- [alpha-D-Glcp-(1-6)-]-alpha-Hepp-(1-5)-R, where R is alpha-Kdop-(2-6)-beta-D-GlcNAcp-(1-6)-D-GlcNAcol in 1 and 4,8-anhydro-Kdool in 2, and Hep is L-glycero-D-manno-heptose. In lipopolysaccharide, the terminal residue of alpha-D-glucosamine possessed a free amino group, as proved by deamination with nitrous acid and the 1H-NMR spectrum of de-O-acylated lipopolysaccharide. The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simulations combined with restrained calculations of side chains based on experimentally determined proton-coupling constants. These calculations, confirmed by NOE data, displayed several long-range interactions, which resulted in a well-defined three-dimensional structure of the core oligosaccharide.

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Glycosyltransferases acting on O-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes and O-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Gal beta (1-3)GalNAc alpha Bzl (1, core 1), GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha Bzl (2, core 2), GlcNAc beta (1-3)GalNAc alpha Bzl (3, core 3), GlcNAc beta (1-6)[GlcNAc beta (1-3)]GalNAc alpha Bzl (4, core 4), GlcNAc beta (1-6)GalNAc alpha Bzl (5, core 6), the elongated core 2, Gal beta (1-4)GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha pNp (6) and GalNAc alpha-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1-6) linkage is much more flexible than the (1-3) or the (1-4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additional N-acetyl group in (3). The conformational equilibrium of beta (1-3) glycosidic bonds of 1 and 3 was not affected by attaching a beta (1-6) linked GlcNAc unit to the GalNAc residue in 2 and 4. However, experimental and theoretical data show that the beta (1-6) linkages of the trisaccharides 2 and 4, which carry an additional beta (1-3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1-6) linkage of 2, 4, and 6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of approximately 4 kcal mol-1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.

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A Metropolis Monte Carlo (MMC) algorithm was applied to explore conformational spaces spanned by the exocyclic dihedral angles of four disaccharides alpha-D-Man(1-->3)-alpha-D-Man(1-->O)Me (1), alpha-D-Man(1-->2)-alpha-D-Man(1-->O)Me (2), methyl beta-cellobioside (3), and methyl beta-maltoside (4). The simulation method uses the HSEA force field and randomly samples the conformational space with an automatic preference for low-energy states. In comparison to a systematic grid search, MMC offers a much more convenient and efficient protocol for the computation of ensemble average values of experimentally accessible NMR parameters such as NOE effects or 3J coupling constants. Energy barriers of a few kcal/mol were found to be surmounted easily when running the simulations with the temperature parameter set at room temperature, whereas passing significantly higher barriers required elevated temperature parameters. Ensemble average NOE values were calculated using the MMC technique and a conventional systematic grid search showing that the MMC method adequately samples the conformational spaces of 1-4. Theoretical NOEs derived for global or local minimum conformations are different from ensemble average values, and it is shown that averaged NOEs agree significantly better with experimental data. Ensemble average NOEs for 1 derived from MMC/HSEA, and previously reported MM2CARB and AMBER calculations all showed good agreement with experimental data, with MMC/HSEA giving the closest fit.

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We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used [1H,1H] NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear [1H,13C] NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on 13C chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha (2----6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the alpha (2----6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with phi angles at -60 degrees or 180 degrees and of the 3 staggered rotamers of the omega angle with a predominant gt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

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The hemicellulosic polysaccharide xyloglucan binds with a strong affinity to cellulosic cell wall microfibrils, the resulting heterogeneous network constituting up to 50% of the dry weight of the cell wall in dicotyledonous plants. To elucidate the molecular details of this interaction, we have performed theoretical potential energy calculations of the static and dynamic equilibrium conformations of xyloglucan using the GEGOP software. In particular, we have evaluated the preferred sidechain conformations of hexa-, octa-, deca- and heptadecasaccharide model fragments of xyloglucan for molecules with a cellulose-like, flat, glucan backbone, and a cellobiose-like, twisted, glucan backbone conformation. For the flat backbone conformation the determination of static equilibrium molecular conformations revealed a tendency for sidechains to fold onto one surface of the backbone, defined here as the H1S face, in the fucosylated region of the polymer. This folding produces a molecule that is sterically accessible on the opposite face of the backbone, the H4S face. Typically, this folding onto the H1S surface is significantly stabilized by favorable interactions between the fucosylated, trisaccharide sidechain and the backbone, with some stabilization from adjacent terminal xylosyl sidechains. In contrast, the trisaccharide sidechain folds onto the H4S face of xyloglucan fragments with a twisted backbone conformation. Preliminary NMR data on nonasaccharide fragments isolated from sycamore suspension-cultured cell walls are consistent with the hypothesis that the twisted conformation of xyloglucan represents the solution form of this molecule. Metropolis Monte Carlo (MMC) simulations were employed to assess sidechain flexibility of the heptadecasaccharide fragments. Simulations performed on the flat, rigid, backbone xyloglucan indicate that the trisaccharide sidechain is less mobile than the terminal xylosyl sidechains. MMC calculations on a fully relaxed molecule revealed a positive correlation between a specific trisaccharide sidechain orientation and the 'flatness' of the backbone glucosyl residues adjacent to this sidechain. These results suggest that the trisaccharide sidechain may play a role in the formation of nucleation sites that initiate the binding of these regions to cellulose. Based on these conformational preferences we suggest the following model for the binding of xyloglucan to cellulose. Nucleation of a binding site is initiated by the fucosylated, trisaccharide sidechain that flattens out an adjacent region of the xyloglucan backbone. Upon contacting a cellulose microfibril this region spreads by step-wise flattening of successive segments of the backbone. Self-association of xyloglucan molecules in solution may be prevented by the low frequency of formation of these nucleation sites and the geometry of the molecules in solution.

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Radon control systems were installed and evaluated in fourteen homes in the Spokane River Valley/Rathdrum Prairie and in one home in Vancouver, Washington. Because of local soil conditions, subsurface ventilation (SSV) by pressurization was always more effective in these houses than SSV by depressurization in reducing indoor radon levels to below guidelines. Basement overpressurization was successfully applied in five houses with airtight basements where practical-sized fans could develop an overpressure of 1 to 3 Pascals. Crawlspace ventilation was more effective than crawlspace isolation in reducing radon entry from the crawlspace, but had to be used in conjunction with other mitigation techniques, since the houses also had basements. Indoor radon concentrations in two houses with air-to-air heat exchangers (AAHX) were reduced to levels inversely dependent on the new total ventilation rates and were lowered even further in one house where the air distribution system was modified. Sealing penetrations in the below-grade surfaces of substructures was relatively ineffective in controlling radon. Operation of the radon control systems (except for the AAHX's) made no measureable change in ventilation rates or indoor concentrations of other measured pollutants. Installation costs by treated floor area ranged from approximately $4/m2 for sealing to $28/m2 for the AAHX's. Based on the low electric rates for the region, annual operating costs for the active systems were estimated to be approximately $60 to $170.

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Energetically favored conformations of glycopeptide 1 were calculated using the newly developed force-field program, GEGOP (geometry of glycopeptides). The three-dimensional structure of glycopeptide 1, which is part of the Fc fragment of IgG1, has been calculated. 1 contains 27 amino acid residues from Pro291 to Lys317 and a biantennary decasaccharide N-linked to Asn297. The conformations of the peptide and the carbohydrate parts are shown to be mutually dependent. Single glycosyl residues of 1 exhibit interaction energies of up to -31.8 kJ/mol with the peptide portion. Generally, only a few of the glycosyl residues of the oligosaccharide moiety express significant interaction energies with the peptide part. No easy prediction is possible of glycosyl residues which exhibit favorable interaction energies. However, in all of the calculated structures, the glycosyl residues of the 1-6-linked branches show strong attractive forces for the peptide part. 1-6-glycosidically linked branches can adopt a larger number of conformations than other linkages due to their high flexibility which allows more favorable interactions with proteins. We developed the GEGOP program in order to be able to study the preferred conformations of large glycopeptides. The program is based on the GESA (geometry of saccharides) program and utilizes the HSEA (hard sphere exo anomeric) force field for the carbohydrate part and the ECEPP/2 (empirical conformation energy program for peptides) force field [Némethy, G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 87, 1883-1887] for the peptide part. The GEGOP program allows the simultaneous relaxation of all rotational degrees of freedom of these glycoconjugates during the energy optimization process. Thus, mutual interactions between glycosyl and amino acid residues can be studied in detail.

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Research-based procedures for characterizing the causes of elevated indoor 222Rn levels and guiding the selection of an appropriate control technique were evaluated at seven New Jersey houses. Procedures such as thorough visual inspections, blower door air leakage tests, pressure field mapping, subsurface vacuum extension tests, sampling of 222Rn concentrations throughout the substructure, and measurements of the additional depressurization caused by various appliances all were found to furnish important information to the mitigation contractor or researcher. An analysis of data from these and other diagnostic techniques performed at the seven houses also indicated: (1) regions of very high permeability existed directly adjacent to the exterior of substructure walls and floors; (2) the additional substructure depressurization caused by operation of forced-air furnaces and attic exhaust fans could exceed 1 Pascal; (3) 222Rn concentrations below basement slabs and slabs-on-grade adjoining below grade basement walls were approximately seven times higher than those within block wall cavities; and (4) air leakage areas of crawlspace and basement ceilings were quite large, ranging up to 0.15 m2. The pressure field mapping tests identified the areas surrounding the substructure that were well coupled to the indoors. Using flow, pressure difference, and 222Rn concentration data, indices of soil gas entry potential and 222Rn entry potential were developed to indicate the areas of the substructure that may have high entry rates of soil gas and 222Rn, respectively. These indices could be helpful for quantifying the relative resistance to soil gas movement of substructure surfaces and surrounding soils and for determining the placement of 222Rn control systems.

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Fourteen single-family detached houses in Spokane, Washington, and Coeur D'Alene, Idaho, were monitored for two years after high concentrations of indoor radon had been mitigated. Each house was monitored quarterly using mailed alpha-track radon detectors deployed in each zone of the structure. To assess performance of mitigation systems during the second heating season after mitigation, radon concentrations in seven houses were monitored continuously for several weeks, mitigation systems in all houses were inspected, and selected other measurements were taken. In addition, occupants were also interviewed regarding their maintenance, operation, and subjective evaluation of the radon mitigation systems. Quarterly alpha-track measurements showed that radon levels had increased in most of the homes during many follow-up measurement periods when compared with concentrations measured immediately after mitigation. Mitigation-system performance was adversely affected by (1) accumulated outdoor debris blocking the outlets of subsurface pressurization pipes; (2) fans being turned off (e.g., because of excessive noise or vibration); (3) air-to-air heat exchanger, basement pressurization, and subsurface ventilation fans being turned off and fan speeds reduced; and (4) crawl-space vents being closed or sealed.

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A compilation of data from earlier studies of 172 homes in the Pacific Northwest indicated that approximately 65 percent of the 46 homes tested in the Spokane River Valley/Rathdrum Prairie region of eastern Washington/northern Idaho had heating season indoor radon (222Rn) concentrations above the U. S. EPA guideline of 148 Bq m-3 (4 pCi L-1). A subset of 35 homes was selected for additional study. The primary source of indoor radon in the Spokane River Valley/Rathdrum Prairie was pressure-driven flow of soil gas containing moderate radon concentrations (geometric mean concentration of 16,000 Bq m-3) from the highly permeable soils (geometric mean permeability of 5 x 10(-11) m2) surrounding the house substructures. Estimated soil gas entry rates ranged from 0.4 to 39 m3h-1 and 1 percent to 21 percent of total building air infiltration. Radon from other sources, including domestic water supplies and building materials was negligible. In high radon homes, winter indoor levels averaged 13 times higher than summer concentrations, while in low radon homes winter levels averaged only 2.5 times higher. Short-term variations in indoor radon were observed to be dependent upon indoor-outdoor temperature differences, wind speed, and operation of forced-air furnace fans. Forced-air furnace operation, along with leaky return ducts and plenums, and openings between the substructure and upper floors enhanced mixing of radon-laden substructure air throughout the rest of the building.

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Central chemosensitivity is ascribed to three areas on the ventral medullary surface. The discharge frequency of neurons observed in these areas depends upon pH. Other neurons within the same areas do not change their frequency during acidosis or alkalosis. Histologically the areas are characterized by superficial nerve cells.

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