Interrelation between thrombin structure and its stability

Data concerning peculiarities of fermentative nature and structure of thrombin in water-salt solution have been generalized; regularities of stabilizing effect made on thrombin by various polyols and other substances have been analyzed. It has been shown that formation of thrombin optimum macrostructure is one of the methods of its stabilization. Presence of different dissolving additives changes this enzymes hydration and this affects its stability and activity. There exist some systems to stabilize thrombin solutions. The systems consist of various salts, low-molecular and high-molecular polyols, surfactants, protein chain, composition buffer, etc. It has been shown that optimal concentrations of polyols, buffer salts and surfactants, as well as protein interaction increase considerably thrombin stability, preserving secondary structure even under its low concentration in the solution.     

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells

It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.     

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.     

Technical refolding of proteins: Do we have freedom to operate?

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate.     

Carbon dioxide induced silk protein gelation for biomedical applications

We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive β-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

Fusion tails for the recovery and purification of recombinant proteins.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.     

Characterization of mammalian neurofilament subunits by circular dichroism

Critical steps in the disassembly and reassembly of neurofilaments, the intermediate filaments of neurons, have been investigated. Bovine neurofilament subunits (Mr 210 000, 160 000 and 70 000) were purified by urea-polyacrylamide gel electrophoresis and renatured by dialysis against several non-denaturing buffers. The quality of the protein renaturation was measured by circular dichroism. The spectra of renatured neurofilament subunits were interpreted in terms of secondary structure and this showed that the solubilization of proteins in guanidine-HCl buffers is more suitable than in urea buffer for a good recovery of a filamentous structure. Furthermore, it is shown that (i) the three neurofilament subunits exhibit specific CD spectra, with shapes reminiscent of those obtained for the alpha/beta class of proteins and that (ii) there is good correlation between CD spectra, the state of renaturation and the ability of the proteins to assemble into filamentous structures. We conclude that CD studies of neurofilament proteins should help in understanding the numerous variables affecting the disassembly and reassembly of neurofilaments.     

Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel

Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 microl of buffer by a modification of the procedure of Gombocz and Cortez. The electroeluate was freed of SDS, ultrafiltered and subjected to MALDI-TOF mass spectrometry. The masses of the five native proteins were found to be maintained after electrophoresis and electroelution in the presence of the potential contaminants SDS, barbituric acid and Cascade Blue. The procedure of protein transfer from SDS-PAGE into mass spectrometry, without excision of bands, gel maceration and protein recovery by diffusion, therefore is shown to be suitable for the identification by mass of intact proteins derived from gel electrophoretic bands.     

The effect of amphiphilic peptide surfactants on the light-harvesting complex II

The peptide surfactants are amphiphilic peptides which have a hydrophobic tail and a hydrophilic head, and have been reported to stabilize and protect some membrane proteins more effectively than conventional surfactants. The effects of a class of peptide surfactants on the structure and thermal stability of the photosynthetic membrane protein lightharvesting complex II (LHCII) in aqueous media have been investigated. After treatment with the cationic peptide surfactants A₆K, V₆K₂, I₅K₂ and I₅R₂, the absorption at 436 nm and 470 nm decreased and the absorption at 500–510 nm and 684–690 nm increased. Moreover, the circular dichroism (CD) signal intensity in the Soret region also decreased significantly, indicating the conformation of some chlorophyll (Chl) a, Chl b, and the xanthophyll molecules distorted upon cationic peptide surfactants treatment. The anionic peptide surfactants A₆D and V₆D₂ had no obvious effect on the absorption and CD spectra. Except for A₆D, these peptides all decreased the thermal stability of LHCII, indicating that these peptides may reconstitute protein into a less stable conformation. In addition, the cationic peptide surfactants resulted in LHCII aggregation, as shown by sucrose gradient ultracentrifugation and fluorescence spectra.     

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants

The electrophoretic behavior of α-lactalbumin and β-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein–surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the α-lactalbumin and the β-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein–surfactant interactions.     

Integration of Gas Enhanced Oil Recovery in Multiphase Fermentations for the Microbial Production of Fuels and Chemicals

In multiphase fermentations where the product forms a second liquid phase or where solvents are added for product extraction, turbulent conditions disperse the oil phase as droplets. Surface‐active components (SACs) present in the fermentation broth can stabilize the product droplets thus forming an emulsion. Breaking this emulsion increases process complexity and consequently the production cost. In previous works, it has been proposed to promote demulsification of oil/supernatant emulsions in an off‐line batch bubble column operating at low gas flow rate. The aim of this study is to test the performance of this recovery method integrated to a fermentation, allowing for continuous removal of the oil phase. A 500 mL bubble column is successfully integrated with a 2 L reactor during 24 h without affecting cell growth or cell viability. However, higher levels of surfactants and emulsion stability are measured in the integrated system compared to a base case, reducing its capacity for oil recovery. This is related to release of SACs due to cellular stress when circulating through the recovery column. Therefore, it is concluded that the gas bubble‐induced oil recovery method allows for oil separation and cell recycling without compromising fermentation performance; however, tuning of the column parameters considering increased levels of SACs due to cellular stress is required for improving oil recovery.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.     

Effect of carbon dioxide on neonatal mouse lung: a genomic approach.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO(2), but only a few at 12% CO(2). Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO(2) seem to depend on the level to which the lung is exposed.