Methane as a resource: can the methanotrophs add value?

Methane is an abundant gas used in energy recovery systems, heating, and transport. Methanotrophs are bacteria capable of using methane as their sole carbon source. Although intensively researched, the myriad of potential biotechnological applications of methanotrophic bacteria has not been comprehensively discussed in a single review. Methanotrophs can generate single-cell protein, biopolymers, components for nanotechnology applications (surface layers), soluble metabolites (methanol, formaldehyde, organic acids, and ectoine), lipids (biodiesel and health supplements), growth media, and vitamin B12 using methane as their carbon source. They may be genetically engineered to produce new compounds such as carotenoids or farnesene. Some enzymes (dehydrogenases, oxidase, and catalase) are valuable products with high conversion efficiencies and can generate methanol or sequester CO2 as formic acid ex vivo. Live cultures can be used for bioremediation, chemical transformation (propene to propylene oxide), wastewater denitrification, as components of biosensors, or possibly for directly generating electricity. This review demonstrates the potential for methanotrophs and their consortia to generate value while using methane as a carbon source. While there are notable challenges using a low solubility gas as a carbon source, the massive methane resource, and the potential cost savings while sequestering a greenhouse gas, keeps interest piqued in these unique bacteria.

Fate of pathogen indicators in a domestic blend of food waste and wastewater through a two-stage anaerobic digestion system.

Anaerobic digestion is a viable on-site treatment technology for rich organic waste streams such as food waste and blackwater. In contrast to large-scale municipal wastewater treatment plants which are typically located away from the community, the effluent from any type of on-site system is a potential pathogenic hazard because of the intimacy of the system to the community. The native concentrations of the pathogen indicators Escherichia coli, Clostridium perfringens and somatic coliphage were tracked for 30 days under stable operation (organic loading rate (OLR) = 1.8 kgCOD m(-3) day(-1), methane yield = 52% on a chemical oxygen demand (COD) basis) of a two-stage laboratory-scale digester treating a mixture of food waste and blackwater. E. coli numbers were reduced by a factor of 10(6.4) in the thermophilic stage, from 10(7.5±0.3) to 10(1.1±0.1) cfu 100 mL(-1), but regenerated by a factor of 10(4) in the mesophilic stage. Neither the thermophilic nor mesophilic stages had any significant impact on C. perfringens concentrations. Coliphage concentrations were reduced by a factor of 10(1.4) across the two stages. The study shows that anaerobic digestion only reduces pathogen counts marginally but that counts in effluent samples could be readily reduced to below detection limits by filtration through a 0.22 µm membrane, to investigate membrane filtration as a possible sanitation technique.

Current status of inverse agonism at serotonin2A (5-HT2A) and 5-HT2C receptors.

Contemporary receptor theory was developed to account for the existence of constitutive activity, as defined by the presence of receptor signaling in the absence of any ligand. Thus, ligands acting at a constitutively active receptor, can act as agonists, antagonists, and inverse agonists. In vitro studies have also revealed the complexity of ligand/receptor interactions including agonist-directed stimulus trafficking, a finding that has led to multi-active state models of receptor function. Studies with a variety of cell types have established that the serotonin 5-HT(2A) and 5-HT(2C) receptors also demonstrate constitutive activity and inverse agonism. However, until recently, there has been no evidence to suggest that these receptors also demonstrate constitutive activity and hence reveal inverse agonist properties of ligands in vivo. This paper describes our current knowledge of constitutive activity in vitro and then examines the evidence for constitutive activity in vivo. Both the serotonin 5-HT(2A) and 5-HT(2C) receptors are involved in a number of physiological and behavioral functions and are the targets for treatment of schizophrenia, anxiety, weight control, Parkinsonism, and other disorders. The existence of constitutive activity at these receptors in vivo, along with the possibility of inverse agonism, provides new avenues for drug development.

The effect of biomass density on cellulose solubilisation rates.

The aim of this work was to compare the impact of inoculation density on the rate of cellulose hydrolysis by a rumen derived culture with that of a microbial enrichment from an organic waste anaerobic digester. The results showed a linear relationship between the mass of biomass at the start of the first order degradation phase (Xo) and the first order hydrolysis rate (r) for both rumen inoculated and leachate inoculated cellulose digestions and that the slopes of these relationships were not distinguishable. This suggested that differences in the microbial community, media and other environmental factors had a lesser impact on the hydrolysis rate compared to the effect of the number of cells in the system. This could be of great importance to industrial applications of anaerobic digestion technologies as it suggested that if cells densities in the waste treatment digesters could be boosted to match those seen in the rumen, then the rates of the cellulose hydrolysis would rise.

Digestion of waste bananas to generate energy in Australia.

This paper presents results from laboratory studies to measure the methane yield and rate of digestion of reject bananas. These parameters were determined in experiments that took into account the likely configuration of a full-scale plant in the banana growing region of north Queensland. The digestion was conducted in a 200-l reactor using fed-batch operation, relying entirely on the natural microbial consortia on the reject bananas to avoid reliance on external inocula such as sludge, an undesirable material around food packaging facilities. An enrichment culture was first established in a highly buffered 200-l batch digestion unit. The fed-batch digester was then started by exchanging leachate with the mature batch reactor. Under loading conditions of 0.6 kg VS m(-3)d(-1) over 70 days where the average working volume was 160 l, the digester produced 398+/-20 l CH4 kg VS(-1). Increasing the loading rate to 1.6 kg VS m(-3)d(-1) resulted in a reduced methane yield of 210 l CH4 kg VS(-1) over 23 days of operation, with a concomitant accumulation of banana waste in the digester. The leachate at the end of digestion contained over 4000 mg l(-1)K, 200 mg l(-1) N and 75 mg l(-1), levels that exceed acceptable limits for general agricultural irrigation.

Measurement and quantification of sessile and planktonic microbial populations during the anaerobic digestion of cellulose.

Crystalline cellulose was anaerobically degraded using a leachate inoculum derived from simulated municipal solid waste. Bicinchoninic Acid (BCA) protein assays were used to measure the distribution of biomass during cellulose degradation, including the planktonic and sessile biomass fractions. A comparison of sessile and planktonic microbial growth indicated that the microbial growth was dominated by the planktonic fraction with the biofilms accounting for approximately 25% of the population. Additional biomass measurements were conducted to test the reliability of the BCA protein assays. Total microbial growth was inferred from the accumulation and depletion of ammonia nitrogen measured using flow injection analysis. The planktonic biomass was estimated from direct cell counts using light microscopy and the sessile biomass was estimated by analysing the nitrogen content of the separated and washed cellulose pellet. Regression analysis showed good correlations between the measurement pairs representing the total biomass (R2=0.90), planktonic biomass (R2=0.97) and sessile biomass (R2=0.85), supporting the use of protein assays as an indicator of microbial growth in mixed culture environments.

The uptake of anaerobic digestion for the organic fraction of municipal solid waste - Push versus pull factors.

There are thousands of anaerobic digestion facilities worldwide applied to agricultural waste, energy crops and industrial food processing wastes. Yet, centralised anaerobic digestion for the organic fraction of Municipal Solid Waste (OFMSW) is almost exclusively applied in Europe and California where diversion of organics from landfill is enforced by legislation. Even in the EU however, only 5% of OFMSW is currently digested, with most organic waste incinerated. Municipalities elsewhere are reluctant to switch to biological treatment, even when made financially preferable through mechanisms such as landfill taxes. The uptake of OFMSW anaerobic digestion around the world and the legislative and economic drivers where this has occurred are reviewed. The prime reason for lack of uptake in regions with ample economic drivers is a lack of experience of municipalities and environmental regulators in regulating both the facilities and the disposal of treated organic material to land.

Deterministic mechanisms define the long-term anaerobic digestion microbiome and its functionality regardless of the initial microbial community.

The impact of the starting inoculum on long-term anaerobic digestion performance, process functionality and microbial community composition remains unclear. To understand the impact of starting inoculum, active microbial communities from four different full-scale anaerobic digesters were each used to inoculate four continuous lab-scale anaerobic digesters, which were operated identically for 295 days. Digesters were operated at 15 days solid retention time, an organic loading rate of 1 g COD Lr-1 d-1 (75:25 - cellulose:casein) and 37 °C. Results showed that long-term process performance, metabolic rates (hydrolytic, acetogenic, and methanogenic) and microbial community are independent of the inoculum source. Digesters process performance converged after 80 days, while metabolic rates and microbial communities converged after 120-145 days. The convergence of the different microbial communities towards a core-community proves that the deterministic factors (process operational conditions) were a stronger driver than the initial microbial community composition. Indeed, the core-community represented 72% of the relative abundance among the four digesters. Moreover, a number of positive correlations were observed between higher metabolic rates and the relative abundance of specific microbial groups. These correlations showed that both substrate consumers and suppliers trigger higher metabolic rates, expanding the knowledge of the nexus between microorganisms and functionality. Overall, these results support that deterministic factors control microbial communities in bioreactors independently of the inoculum source. Hence, it seems plausible that a desired microbial composition and functionality can be achieved by tuning process operational conditions.

Integrins regulate opioid receptor signaling in trigeminal ganglion neurons.

The binding of integrins to the extracellular matrix results in focal organization of the cytoskeleton and the genesis of intracellular signals that regulate vital neuronal functions. Recent evidence suggests that integrins modulate G-protein-coupled receptor (GPCR) signaling in hippocampal neurons. In this study we evaluated the hypothesis that integrins regulate the mu opioid receptor in rat trigeminal ganglion neurons. For these studies, primary cultures of adult rat trigeminal ganglion neurons were used to demonstrate the colocalization of beta1 and beta3 integrins with mu opioid receptor in caveolin-1-rich membrane fractions, and at focal adhesions sites generated by integrin ligand binding. Furthermore, we show that the mu opioid receptor agonist, DAMGO ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin), inhibits cyclic AMP (cAMP) accumulation in response to prostaglandin E2 (PGE(2)) stimulation in bradykinin-primed, but not unprimed, cultured trigeminal ganglia neurons. Application of soluble GRGDS (Gly-Arg-Gly-Asp-Ser) peptides that bind specific integrins (i.e. RGD-binding integrins) completely abolished the DAMGO effect in bradykinin-primed trigeminal ganglia neurons, but did not alter bradykinin-mediated hydrolysis of phosphatidylinositol. Likewise, monospecific anti-beta1 and anti-beta3 integrin subunit antibodies blocked this DAMGO effect in bradykinin-primed trigeminal ganglia neurons. Indeed, application of anti-beta1 integrin subunit actually reversed DAMGO signaling, resulting in increased cAMP accumulation in these cells. This suggests that the relative amounts of specific activated integrins at focal adhesions may govern signaling by the mu opioid receptor, perhaps by altering interactions with G proteins (e.g. Galphai vs. Galphas). Collectively, these data provide the first evidence that specific integrins regulate opioid receptor signaling in sensory neurons.

A methanotroph-based biorefinery: Potential scenarios for generating multiple products from a single fermentation.

Methane, a carbon source for methanotrophic bacteria, is the principal component of natural gas and is produced during anaerobic digestion of organic matter (biogas). Methanotrophs are a viable source of single cell protein (feed supplement) and can produce various products, since they accumulate osmolytes (e.g. ectoine, sucrose), phospholipids (potential biofuels) and biopolymers (polyhydroxybutyrate, glycogen), among others. Other cell components, such as surface layers, metal chelating proteins (methanobactin), enzymes (methane monooxygenase) or heterologous proteins hold promise as future products. Here, scenarios are presented where ectoine, polyhydroxybutyrate or protein G are synthesised as the primary product, in conjunction with a variety of ancillary products that could enhance process viability. Single or dual-stage processes and volumetric requirements for bioreactors are discussed, in terms of an annual biomass output of 1000 tonnesyear(-1). Product yields are discussed in relation to methane and oxygen consumption and organic waste generation.

Semi-aerobic fermentation as a novel pre-treatment to obtain VFA and increase methane yield from primary sludge.

There is a growing trend to consider organic wastes as potential sources of renewable energy and value-add products. Fermentation products have emerged as attractive value-add option due to relative easy production and broad application range. However, pre-fermentation and extraction of soluble products may impact down-stream treatment processes, particularly energy recovery by anaerobic digestion. This paper investigates primary sludge pre-fermentation at different temperatures (20, 37, 55, and 70°C), treatment times (12, 24, 48, and 72h), and oxygen availability (semi-aerobic, anaerobic); and its impact on anaerobic digestion. Pre-fermentation at 20 and 37°C succeeded for VFA production with acetate and propionate being major products. Pre-fermentation at 37, 55, and 70°C resulted in higher solubilisation yield but it reduced sludge methane potential by 20%. Under semi-aerobic conditions, pre-fermentation allowed both VFA recovery (43gCODVFAkg(-1)VS) and improved methane potential. The latter phenomenon was linked to fungi that colonised the sludge top layer during pre-fermentation.

Effect of biomass concentration on methane oxidation activity using mature compost and graphite granules as substrata.

Reported methane oxidation activity (MOA) varies widely for common landfill cover materials. Variation is expected due to differences in surface area, the composition of the substratum and culturing conditions. MOA per methanotrophic cell has been calculated in the study of natural systems such as lake sediments to examine the inherent conditions for methanotrophic activity. In this study, biomass normalised MOA (i.e., MOA per methanotophic cell) was measured on stabilised compost, a commonly used cover in landfills, and on graphite granules, an inert substratum widely used in microbial electrosynthesis studies. After initially enriching methanotrophs on both substrata, biomass normalised MOA was quantified under excess oxygen and limiting methane conditions in 160ml serum vials on both substrata and blends of the substrata. Biomass concentration was measured using the bicinchoninic acid assay for microbial protein. The biomass normalised MOA was consistent across all compost-to-graphite granules blends, but varied with time, reflecting the growth phase of the microorganisms. The biomass normalised MOA ranged from 0.069±0.006µmol CH4/mg dry biomass/h during active growth, to 0.024±0.001µmol CH4/mg dry biomass/h for established biofilms regardless of the substrata employed, indicating the substrata were equally effective in terms of inherent composition. The correlation of MOA with biomass is consistent with studies on methanotrophic activity in natural systems, but biomass normalised MOA varies by over 5 orders of magnitude between studies. This is partially due to different methods being used to quantify biomass, such as pmoA gene quantification and the culture dependent Most Probable Number method, but also indicates that long term exposure of materials to a supply of methane in an aerobic environment, as can occur in natural systems, leads to the enrichment and adaptation of types suitable for those conditions.

The elusive nature of intrinsic efficacy.

In the discipline of pharmacology, drugs (ligands) are used as tools to elucidate the processes of biological systems. Because of this, pharmacologists strive to delineate all characteristics of drugs. Decades of research have resulted in the proposal that ligands possess two properties that are intrinsic to the ligand and are invariant of the system in which their effects are investigated. These properties are affinity (the capacity of a drug to bind to a receptor) and intrinsic efficacy (the capacity of a drug to activate or inactivate a receptor). Although affinity is a relatively easy parameter to measure with a variety of techniques, ways of quantifying intrinsic efficacy have remained elusive ever since its inception. Furthermore, recent evidence suggests that intrinsic efficacy might not be a single, ligand-dependent parameter but that agonists might have multiple intrinsic efficacies. William Clarke and Richard Bond discuss several reasons why the claim that intrinsic efficacy is a ligand-dependent parameter should be questioned, and the possible impact of these findings.

Evaluation by respirometry of the degradability of retort water using a shale ash and overburden packed column.

Oil shale processing produces an aqueous wastewater stream known as retort water. The fate of the organic content of retort water from the Stuart oil shale project (Gladstone, Queensland) is examined in a proposed packed bed treatment system consisting of a 1:1 mixture of residual shale from the retorting process and mining overburden. The retort water had a neutral pH and an average unfiltered TOC of 2,900 mg 1(-1). The inorganic composition of the retort water was dominated by NH4+. Only 40% of the total organic carbon (TOC) in the retort water was identifiable, and this was dominated by carboxylic acids. In addition to monitoring influent and effluent TOC concentrations, CO2 evolution was monitored on line by continuous measurements of headspace concentrations and air flow rates. The column was run for 64 days before it blocked and was dismantled for analysis. Over 98% of the TOC was removed from the retort water. Respirometry measurements were confounded by CO2 production from inorganic sources. Based on predictions with the chemical equilibrium package PHREEQE, approximately 15% of the total CO2 production arose from the reaction of NH4+ with carbonates. The balance of the CO2 production accounted for at least 80% of the carbon removed from the retort water. Direct measurements of solid organic carbon showed that approximately 20% of the influent carbon was held-up in the top 20cm of the column. Less than 20% of this held-up carbon was present as either biomass or as adsorbed species. Therefore, the column was ultimately blocked by either extracellular polymeric substances or by a sludge that had precipitated out of the retort water.

Fluctuation of dissolved heavy metal concentrations in the leachate from anaerobic digestion of municipal solid waste in commercial scale landfill bioreactors: The effect of pH and associated mechanisms.

Heavy metals present in landfill leachate have infrequently been related to complete anaerobic degradation municipal solid waste (MSW) due to discrete ages of deposited MSW layers and leachate channelling in landfills. In this study, anaerobic digestion of MSW was performed in two enclosed 1000 tonne bioreactors using a unique flood and drain process. Leachates were characterised in terms of pH, soluble chemical oxygen demand, volatile fatty acids (VFAs), ammonium nitrogen and heavy metals over the entire course of digestion. All parameters, including pH, fluctuated during acidogenesis, acetogenesis and methanogenesis, which strongly impacted on the dynamics of dissolved heavy metal concentrations. The simulation of dissolution and precipitation processes indicated that metal sulphide precipitation was not a factor as metal concentrations exceeded solubility limits. The correlation of pH and dissolved heavy metal concentrations indicated that other, mechanisms were involved in the homogenised conditions within the bioreactors. Beside dissolution and precipitation, the main processes most likely involved in metal distributions were adsorption (Zn, Cu, Ni, Pb and Cd), complexation (Cr) or combinations of both process (As and Co).

The influence of blinding, olfactory bulbectomy, and pinealectomy on twenty four-hour plasma prolactin levels in normal and neonatally androgenized female rats.

The influence of blinding (BLD), olfactory bulbectomy (ANOS), and pinealectomy (PX) on 24-h plasma PRL levels was examined in normal and neonatally androgenized (NA) female rats. NA pups were treated at 3 days of age with 1.25 mg testosterone propionate. Surgery was performed on both NA and normal animals at 22-25 days of age. At 10 weeks of age, animals were vaginally smeared, and at 15 weeks, they were ovariectomized, injected with 0.5 mg polyestradiol phosphate, and fitted with atrial catheters for blood sampling. At 16 weeks of age, blood samples were taken over a 24-h period. In normal animals, BLD plus ANOS resulted in half of the animals exhibiting a diestrous vaginal smear, while the other half exhibited at least one vaginal estrus during the 7-day period when vaginal smears were obtained. Ovarian, oviductal, and uterine weights for blinded (BLD) plus olfactory bulbectomized (ANOS) animals exhibiting diestrous smears were significantly less than those of BLD plus ANOS animals exhibiting periodic vaginal estrus. Plasma PRL levels were lower in both animal groups compared with those in sham controls, but BLD plus ANOS animals with small ovaries had significantly lower plasma PRL and BLD plus ANOS animals with large ovaries. PX of BLD plus ANOS animals resulted in endocrine organ weights comparable to those in controls and elevated the plasma PRL levels to those observed in BLD plus ANOS animals with large ovaries. The PRL levels in BLD, ANOS, and pinealectomized animals, however, were still below those in sham control animals. Sensory deprived animals had peak PRL values at various times of day, and regrouping of the data from the peak PRL time for each animal suggested the presence of a free-running plasma PRL rhythm. In NA animals, a decrease was also observed in endocrine organ weights and plasma PRL for BLD plus ANOS animals similar to that observed for normal BLD plus ANOS animals. However, all animals in the NA BLD plus ANOS group responded with a constant diestrous smear, small ovaries, and low plasma PRL values. PX prevented the decrease in endocrine organ weights and plasma PRL observed in BLD plus ANOS animals. As in normal animals, NA animals also exhibited a free-running PRL rhythm when sensory deprived.(ABSTRACT TRUNCATED AT 400 WORDS)

RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.

1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.

Interactions between effectors linked to serotonin receptors.

In general, there are two types of interactions between effector signaling pathways. "Homologous" interactions are those that occur within a receptor system to alter its own responsiveness, for example the loss of responsiveness (desensitization) that can occur upon agonist occupancy of a receptor. "Heterologous" interactions are those that occur between different receptor systems where the responsiveness of one receptor system is regulated (positively or negatively) by activation of another receptor system (i.e., "cross-talk"). Many, if not all receptors, couple to multiple cellular effector pathways and alterations in the responsiveness of a receptor system can be effector pathway-dependent which underscores the importance of studying each effector coupled to a receptor. Regulation of receptor system responsiveness, and consequently the efficacy of drugs, is a highly dynamic process. Perhaps by exploiting these interactions, new targets for pharmacotherapy may be uncovered which will provide for increased efficacy and specificity of drug action.

Pleiotropic behavior of 5-HT2A and 5-HT2C receptor agonists.

There is now considerable evidence that a single receptor subtype can couple to multiple effector pathways within a cell. Recently, Kenakin proposed a new concept, termed "agonist-directed trafficking of receptor stimulus", that suggests that agonists may be able to selectively activate a subset of multiple signaling pathways coupled to a single receptor subtype. 5-HT2A and 5-HT2C receptors couple to phospholipase C-(PLC) mediated inositol phosphate (IP) accumulation and PLA2-mediated arachidonic acid (AA) release. Relative efficacies of agonists (referenced to 5-HT) differed depending upon whether IP accumulation or AA release was measured. For the 5-HT2C receptor system, some agonists (e.g. TFMPP) preferentially activated the PLC-IP pathway, whereas others (e.g. LSD) favored PLA2-AA. As expected, EC50's of agonists did not differ between pathways. For the 5-HT2A receptor system, all agonists tested had greater relative efficacy for PLA2-AA than for PLC-IP. In contrast, relative efficacies were not different for 5-HT2A agonists when sequential effects in a pathway were measured (IP accumulation vs. calcium mobilization). These data strongly support the agonist-directed trafficking hypothesis.

A university-staffed, private hospital-based air transport service. The initial two-year experience.

During the first two years of a cooperative effort between the University of Texas Medical School at Houston and Hermann Hospital, Houston, a program designed to extend the emergency center to the patient by helicopter treated and moved 1,702 patients. A physician and flight nurse attended patients on each mission. Of all flights, 68.3% were because of major multiple trauma and 28.8% were to the scene of an accident. The magnitude of these injuries was reflected by a mortality of 11% at the scene of the accident and 7% in the emergency room of those transported. The primary purpose of the program is to minimize the time between the catastrophic event and the institution of appropriate medical therapy. Our experience with this program of early stabilization and rapid transport has led to the following observations: (1) single-organ injury is virtually nonexistent in the patient with multiple trauma; (2) clotting abnormalities, even disseminated intravascular coagulopathy, are seen regularly in patients with apparently isolated head injuries; and (3) patients with apparently isolated head injuries often have serious pulmonary function abnormalities.