Amino Acid Specific Stable Nitrogen Isotope Values in Avian Tissues: Insights from Captive American Kestrels and Wild Herring Gulls.

Through laboratory and field studies, the utility of amino acid compound-specific nitrogen isotope analysis (AA-CSIA) in avian studies is investigated. Captive American kestrels (Falco sparverius) were fed an isotopically characterized diet and patterns in δ15N values of amino acids (AAs) were compared to those in their tissues (muscle and red blood cells) and food. Based upon nitrogen isotope discrimination between diet and kestrel tissues, AAs could mostly be categorized as source AAs (retaining baseline δ15N values) and trophic AAs (showing 15N enrichment). Trophic discrimination factors based upon the source (phenylalanine, Phe) and trophic (glutamic acid, Glu) AAs were 4.1 (muscle) and 5.4 (red blood cells), lower than those reported for metazoan invertebrates. In a field study involving omnivorous herring gulls (Larus argentatus smithsonianus), egg AA isotopic patterns largely retained those observed in the laying female's tissues (muscle, red blood cells, and liver). Realistic estimates of gull trophic position were obtained using bird Glu and Phe δ15N values combined with ß values (difference in Glu and Phe δ15N in primary producers) for aquatic and terrestrial food webs. Egg fatty acids were used to weight ß values for proportions of aquatic and terrestrial food in gull diets. This novel approach can be applied to generalist species that feed across ecosystem boundaries.

Whole exome sequencing reveals a novel de novo FOXC1 mutation in a patient with unrecognized Axenfeld-Rieger syndrome and glaucoma.

We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld-Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14 years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss. After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation. Since loss-of-function mutations in FOXC1 are associated with Axenfeld-Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld-Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.

A role for the urokinase-type plasminogen activator system in amyotrophic lateral sclerosis.

There is substantial evidence, implicating extracellular matrix (ECM) regulating enzymes in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The most important ECM-degrading proteases are serine proteases (plasminogen activators, PA) and matrix metalloproteinases (MMPs). Since the role of MMPs in ALS has been addressed recently, we investigated the expression of the serine protease urokinase-type plasminogen activator (uPA) and its receptor in ALS. Employing rtPCR, zymography and immunohistochemistry we analyzed the expression of uPA and its receptor uPAR in spinal cord tissue of ALS cases and in the G93A SOD1 transgenic mouse. In the ventral horn of the spinal cord of ALS cases we found increased uPAR staining of motor neurons. In G93A mice, the expression profile of uPA and uPAR mRNA was significantly increased starting at the age of 90 days as compared to non-transgenic littermates. The uPA-dependent plasminogen activation in G93A mice at endstage increased markedly compared with controls and immunostaining of the spinal cord from G93A mice revealed increased uPAR immunostaining in neurons. To determine the functional role of uPA, we investigated the effect of intraperitoneal (i.p.) administration of the uPA inhibitor WX-340 (10 mg/kg), starting at the age of 30 days (n=18). Treatment with WX-340 prolonged (p<0.05) survival of the animals (135+/-2 vs. 126+/-3) as well as improving rotarod performance. Our experiments demonstrate that uPA and its receptor are expressed in ALS patients and in an animal model of ALS. Early inhibition with a synthetic uPA inhibitor prolonged the life of the transgenic animals. These findings indicate that the urokinase-type plasminogen activator system may play a role in the complex pathogenesis of ALS.

Evaluation of cerebrospinal fluid uPA, PAI-1, and soluble uPAR levels in HIV-infected patients.

To evaluate the potential role of the uPAR/uPA/PAI-1 system in HIV-induced blood-brain-barrier (BBB) disruption, CSF uPA-dependent plasminogen activation (PdPA) was analyzed by casein zymography, and CSF protein levels of all three molecules were measured by ELISA. CSF uPAR, but not uPA, PAI-1, or PdPA levels was significantly increased in neurologically compromised HIV+ patients. Only individual patients with severe AIDS dementia complex had increased levels of uPA (but not PAI-1) which fell upon initiation of antiretroviral therapy. The levels of all three molecules did not correlate with the CSF to serum albumin ratio suggesting not an important role in HIV-induced BBB disruption.

The origin of organic matter in the Martian meteorite ALH84001.

Stable carbon isotope measurements of the organic matter associated with the carbonate globules and the bulk matrix material in the ALH84001 Martian meteorite indicate that two distinct sources are present in the sample. The delta 13C values for the organic matter associated with the carbonate globules averaged -26% and is attributed to terrestrial contamination. In contrast, the delta 13C values for the organic matter associated with the bulk matrix material yielded a value of -15%. The only common sources of carbon on the Earth that yield similar delta 13C values, other then some diagenetically altered marine carbonates, are C4 plants. A delta 13C value of -15%, on the other hand, is consistent with a kerogen-like component, the most ubiquitous form of organic matter found in carbonaceous chondrites such as the Murchison meteorite. Examination of the carbonate globules and bulk matrix material using laser desorption mass spectrometry (LDMS) indicates the presence of a high molecular weight organic component which appears to be extraterrestrial in origin, possibly derived from the exogenous delivery, of meteoritic or cometary debris to the surface of Mars.

The origin of organic matter in the Martian meteorite ALH84001.

Stable carbon isotope measurements of the organic matter associated with the carbonate globules and the bulk matrix material in the ALH84001 Martian meteorite indicate that two distinct sources are present in the sample. The delta 13C values for the organic matter associated with the carbonate globules averaged -26% and is attributed to terrestrial contamination. In contrast, the delta 13C values for the organic matter associated with the bulk matrix material yielded a value of -15%. The only common carbon sources on the Earth that yield similar delta 13C values, other then some diagenetically altered marine carbonates, are C4 plants. A delta 13C value of -15%, on the other hand, is consistent with a kerogen-like component, the most ubiquitous form of organic matter found in carbonaceous chondrites such as the Murchison meteorite. Examination of the carbonate globules and bulk matrix material using laser desorption mass spectrometry (LDMS) indicates the presence of a high molecular weight organic component which appears to be extraterrestrial in origin, possibly derived from the exogenous delivery of meteoritic or cometary debris to the surface of Mars.

Consistent fractionation of 13C in nature and in the laboratory: growth-rate effects in some haptophyte algae.

The carbon isotopic fractionation accompanying formation of biomass by alkenone-producing algae in natural marine environments varies systematically with the concentration of dissolved phosphate. Specifically, if the fractionation is expressed by epsilon p approximately delta e - delta p, where delta e and delta p are the delta 13C values for dissolved CO2 and for algal biomass (determined by isotopic analysis of C37 alkadienones), respectively, and if Ce is the concentration of dissolved CO2, micromole kg-1, then b = 38 + 160*[PO4], where [PO4] is the concentration of dissolved phosphate, microM, and b = (25 - epsilon p)Ce. The correlation found between b and [PO4] is due to effects linking nutrient levels to growth rates and cellular carbon budgets for alkenone-containing algae, most likely by trace-metal limitations on algal growth. The relationship reported here is characteristic of 39 samples (r2 = 0.95) from the Santa Monica Basin (six different times during the annual cycle), the equatorial Pacific (boreal spring and fall cruises as well as during an iron-enrichment experiment), and the Peru upwelling zone. Points representative of samples from the Sargasso Sea ([PO4] < or = 0.1 microM) fall above the b = f[PO4] line. Analysis of correlations expected between mu (growth rate), epsilon p, and Ce shows that, for our entire data set, most variations in epsilon p result from variations in mu rather than Ce. Accordingly, before concentrations of dissolved CO2 can be estimated from isotopic fractionations, some means of accounting for variations in growth rate must be found, perhaps by drawing on relationships between [PO4] and Cd/Ca ratios in shells of planktonic foraminifera.

Study of structure and function of recombinant pea root plastid porin by biophysical methods.

Pea root plastid porin (Fischer et al. (1994) J. Biol. Chem. 269, 25754-25760), which belongs to the family of mitochondrial (eukaryotic) porins, was expressed in Escherichia coli in high amounts using the pQE expression system. The recombinant protein was reconstituted into lipid bilayer membranes, and its characteristic properties were compared to those of the native porin isolated from pea root plastids. No significant difference was found between the native and the recombinant form when the protein was preincubated in detergent and sterol. The recombinant porin seems to be a valuable model system for the study of eukaryotic porins by spectroscopic methods, in which high amounts of protein are needed. CD spectroscopy was performed to determine the secondary structure of the porin under different conditions. It was found to have a high degree of beta-sheet structure in the nonionic detergent Genapol X-80 and in lipid vesicles. The more polar detergent sodium dodecyl sulfate (SDS) induced a large amount of alpha-helix structure in the protein. Addition of sterol to the porin in Genapol buffer did not influence its secondary structure to any measurable extent, whereas it had a strong influence on channel forming activity in black lipid bilayers. First refolding experiments performed in decreasing urea concentrations are discussed together with the results of the other measurements with regard to protein folding and channel formation.

Isolation of mitochondrial porin of the fly Protophormia: porin modification by the pesticide CGA 140'408 studied in lipid bilayer membranes.

Mitochondrial porin from the fly Protophormia was solubilized with detergent from whole mitochondria and purified by chromatography across a hydroxyapatite (HPT) column. The purified protein had an apparent molecular mass of about 30 kDa on SDS-PAGE. Partial sequencing of the protein confirmed that it is porin. When reconstituted in planar lipid bilayer membranes, porin formed ion-permeable channels with single-channel conductances of 2.4 and 4.5 nS in 1 M KCl. At low voltage, Protophormia porin displayed the properties of a general diffusion pore and had a small selectivity for anions over cations. At transmembrane potentials starting with about 20-30 mV, the channel switched in closed state, which is still ion-permeable. Our results suggest that Protophormia porin possesses functional properties similar to those of other mitochondrial porins. Porin was also isolated and purified from mitochondria, which were treated with the carbodiimide CGA 140'408 It represents the active derivative of diafenthiuron a new acaricide and insecticide. This carbodiimide labels both a F0-component of the inner membrane ATPase and outer membrane porin in a similar way as N,N'-dicyclohexylcarbodiimide (DCCD). Reconstitution experiments with the CGA 140'408-modified porin showed no significant effect of the modification on the single-channel conductance, suggesting that CGA 140'408 binds outside the channel. The voltage-dependence of the CGA 140'408-modified porin was changed with respect to the unmodified form. The closed configuration of the pesticide-modified channel was reached at smaller transmembrane potentials, suggesting a shift of the open to the closed state of Protophormia porin by pesticide binding. A possible contribution of this effect to the pesticide action is discussed.

The role of the N and C termini of recombinant Neurospora mitochondrial porin in channel formation and voltage-dependent gating.

To investigate the role of the N and C termini in channel function and voltage-dependent gating of mitochondrial porin, we expressed wild-type and mutant porins from Neurospora crassa as His-tag fusion products in Escherichia coli. Large quantities of the proteins were purified by chromatography across a nickle-nitrilotriacetic acid-agarose column under denaturing conditions. The purified His-tagged wild-type protein could be functionally reconstituted in the presence of detergent and sterol and behaved in black lipid bilayer membranes indistinguishably from native porin isolated from Neurospora crassa mitochondria. Mutants of porin lacking part of the N terminus (DeltaN2-12porin, DeltaN3-20porin), part of the C terminus (DeltaC269-283porin), or both (DeltaN2-12/DeltaC269-283porin) also showed channel forming activity. The mutant porin lacking the C terminus had a smaller single channel conductance than the wild-type protein, but its other biophysical properties were identical. DeltaN2-12porin and DeltaN3-20porin formed noisy channels with decreased channel stability. These channels were still voltage-dependent. DeltaN2-12/DeltaC269-283porin lost channel stability and had altered gating characteristics. These results are discussed with respect to different models that have been proposed in the literature for the structure of mitochondrial porin channels.

The role of sterols in the functional reconstitution of water-soluble mitochondrial porins from plants.

Water-soluble porins were prepared from native mitochondrial porins isolated from different plants (pea and corn). In the water-soluble form the porins have lost their channel-forming properties. The water-soluble porins were investigated for the influence of different sterols on their membrane activity and their channel-forming properties in lipid bilayer membranes. Our experiments demonstrated that the water-soluble porins regained channel forming activity when the protein was preincubated with different sterols in the presence of a detergent. The channels formed in lipid bilayer membranes after this procedure regain in many but not all cases the original properties of the native mitochondrial porins. Preincubation with other sterols led to a change in the single-channel conductance or to a complete loss of the voltage dependence. The sterols had also a strong influence on the channel-forming activity of the porins. Preincubation of water-soluble pea porin with the plant sterol beta-sitosterol resulted in a considerable higher channel-forming activity than with all the other sterols used for preincubation. The role of the sterols in the channel-forming complex is discussed.

Lead content of plants and soils from three abandoned smelter areas in and near Socorro, New Mexico.

Two primary lead smelters and one secondary lead smelter have been active in the Socorro, New Mexico (USA) area in the last 110 years: the Billing smelter from 1883 to 1894, the Cuba Road smelter from 1881 to 1900, and Cal West from 1979 to 1984. Samples of plants and surface soil under each plant from all three sites were analysed for lead. The plants consisted of sparse grasses, cacti, creosote bush, snakeweed, mesquite and fourwing saltbush. Lead levels in the plants increased (2-440 µg g(-1)) as the lead in the alkaline soils (25-10000 ng g(-1)) increased. However, the BAC (biological absorption coefficient), which is the ratio of lead content in the plant to the lead content in the soil, a measure of relative accumulation, decreased by one to two orders of magnitude, except for grasses and snakeweed. At background lead levels, there was little difference between lead in rootsversus foliage. At high lead levels, there was higher lead in roots versus foliage at the Billing and Cuba Road sites. The reverse was noted at Cal West. Because this is a recent operation, the higher lead in foliage may be due to foliar uptake. Plant growth at all sites appeared healthy.

Effect of anesthetic variables on dermatomal somatosensory-evoked potential monitoring in elective lumbar spinal surgery.

We studied 108 adult cases of elective lumbar surgery using dermatomal somatosensory-evoked potential (DSEP) monitoring to evaluate its usefulness due to concern over potential neurologic injury during pedicle screw insertion. Both surgeons used all of the necessary precautions required during surgery so that DSEP monitoring was not the "primary," but rather a backup system for operative security. Quality tracings were obtained in 71% of cases; anesthetic difficulties being the major cause of poor monitoring. There were no neurological complications related to pedicle screw insertion. We found that DSEP monitoring was an excellent method to verify intraoperative neurological status, but required a high degree of cooperation between the anesthesiologists, monitoring technician, and surgeons. In today's cost-containment environment, its usefulness is subjected to the expertise of the spine surgeon and the hospital setting.

Opioid pharmacotherapy in the management of cancer pain: a survey of strategies used by pain physicians for the selection of analgesic drugs and routes of administration.

BACKGROUND: This survey documents the strategies used by pain control physicians in the selection of opioid drugs and routes of administration in the management of inpatients referred to a cancer pain service. METHODS: The following approaches were prospectively evaluated during the treatment of 100 consecutive inpatients: 1) the influence of the evaluation of the goals of care on decision making, 2) selection of opioid drugs, 3) indications for changing opioid drugs and the frequency with which this strategy is used, and 4) selection of route of administration. RESULTS: Eighty of the 100 patients underwent a total of 182 changes in drug, route, or both drug and route before discharge or death. The major reasons for change were to improve the convenience of treatment regimen in the setting of adequate pain relief (31.4%), diminish side effects in the setting of controlled pain (25.0%), reduce the invasiveness of therapy in the setting of controlled pain (19.3%), and simultaneously improve pain control and reduce opioid toxicity (17.7%). When opioid toxicity was the reason for change, physicians changed the opioid drug in 71% of cases and the route in 29%. When convenience or invasiveness were targeted, the physicians changed the route in 61% of cases and the opioid in 39%. Forty-four patients required one or more change in the opioid, and 20 required 2 or more changes (range, 2-6 changes). At the time of discharge (n = 82), morphine was more commonly selected than hydromorphone or fentanyl (39% vs. 23% vs. 17%) and the routes of administration were oral (57%), transdermal (18%), intravenous (18%), subcutaneous (5%), and intraspinal (4%). Therapeutic changes were associated with improvement in physician-recorded pain intensity and a lower prevalence of cognitive impairment, hallucinations, nausea and vomiting, and myoclonus among patients who were discharged from the hospital. CONCLUSIONS: These data illustrate the application of strategies for selections of opioid drugs and their route of administration that are recommended in current guidelines for the management of cancer pain.

Role of sterols in the functional reconstitution of water-soluble mitochondrial porins from different organisms.

Experiments were performed on lipid bilayer membranes with water-soluble mitochondrial porins from different eukaryotic organisms, such as Dictyostelium discoideum, Paramecium, and rat liver, to study the requirements of functional reconstitution of the porins. The water-soluble porins lost their associated lipids and sterols and are unable to form channels in lipid bilayer membranes. We demonstrate that the water-soluble porins regain their channel-forming ability after preincubation of the polypeptides with sterols in the presence of detergents. Mitochondrial porin from Dictyostelium discoideum maintained after this procedure its original properties, in particular the voltage dependence. Water-soluble mitochondrial porins from Paramecium tetraurelia and from rat liver were also activated upon preincubation with different sterols in detergent but showed voltage-dependences that were different from those of detergent-purified porins. Furthermore, the voltage dependence depended on the sterol used for preincubation. Interestingly, the preincubation with sterols can likewise be used to activate detergent-purified mitochondrial porins that may have lost associated sterol during isolation and purification procedures.

Porins from plants. Molecular cloning and functional characterization of two new members of the porin family.

Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.

Porphyrin and chlorin distributions in a Late Pliocene lacustrine sediment.

The tetrapyrroles in a highly immature Late Pliocene lacustrine sediment (Willershausen, Germany) show a simple distribution of both chlorin and porphyrin components as the free bases. The major components are C32 desoxophylloerythroaetioporphyrin (DPEP), a C33 bicycloalkano porphyrin, the chlorin analogue of the latter, and desoxophylloerythrin and its chlorin counterpart. The structure of the novel bicycloalkano chlorin was determined using a combination of two-dimensional phase-sensitive COSY NMR and nOe studies. Measurements of delta 13C and other data indicate that DPEP and the bicycloalkano porphyrin were derived from the chlorophyll(s) of photosynthetic organisms utilising a common source of CO2, probably diatoms. The occurrence of DPEP and other minor alkyl porphyrins indicates that the chlorophyll defunctionalisation pathway leading to these components can occur at low temperature and was probably biologically mediated, as was the condensation leading to the fused ring components.

Isotopic biogeochemistry of the Oxford Clay Formation (Jurassic), UK.

A total of 165 samples was obtained from the Oxford Clay Formation at seven different sites. Nearly all were from the Peterborough Member (Lower Oxford Clay), but seven were from the Stewartby and Weymouth Members (Middle and Upper Oxford Clay respectively). Five samples from the underlying Kellaways Formation were also examined. Stratigraphic relationships were estimated on the basis of ammonite subzones and results from all locations can be placed along a single stratigraphic scale. The following were determined for all samples: abundance and isotopic composition of organic carbon, abundances of carbonate carbon and total sulphur, and the Rock-Eval pyrolysis parameters hydrogen index, oxygen index and Tmax. For a subset of eight samples selected to be representative of geochemical and apparent palaeoenvironmental variations, soluble organic compounds were extracted and the isotopic composition of pristane, phytane, and long-chain n-alkanes determined by isotope-ratio-monitoring gas chromatograph mass spectrometry. Concentrations of organic carbon in samples from the Peterborough Member ranged from 0.5 to 16.6 % and delta values of total organic carbon (TOC) ranged from -27.7 to -23.1% v. PDB. Shales dominated by epifaunal bivalve assemblages have high concentrations of TOC and values of H index approaching 800, indicating preservation of hydrogen-rich organic material. Conversely, shell beds and calcareous and silty clay beds have lower abundances of TOC and values of H index dropping below 100, indicating extensive oxidation of the organic matter. Isotopic composition of pristane and phytane in the Peterborough and Stewartby Members average -31.7%, those in the Weymouth Member average -29.8. Values of delta for long-chain n-alkanes average -28%. Together these results indicate delta values for primary inputs as follows: terrestrial vascular plants, -23.5%; Peterborough Member algae, -28.2; Stewartby Member algae, -29.1%; Weymouth Member algae, -26.6% Comparison of primary delta values to those of TOC indicates that in some cases secondary processes enriched TOC relative to primary inputs by as much as 4%. Paleontological evidence in these same beds indicates development of extensive food-webs and supports attribution of this isotopic enrichment to heterotrophic reworking.

Microscale determination of the spectral characteristics and carbon-isotopic compositions of porphyrins.

Molar extinction coefficients for band III of Ni porphyrins are calculated from results of spectrophotometric and manometric analyses of individual etioporphyrins, DPEP, cyclic, and diDPEP porphyrins known to initially be pure from mass spectrometry, 1H NMR, and analytical HPLC studies. A method for determining carbon-isotopic compositions and purity of micromolar quantities of individual porphyrins using combined spectrophotometric and manometric techniques is presented.

Carbon and nitrogen isotopic compositions of alkyl porphyrins from the Triassic Serpiano oil shale.

The carbon and nitrogen isotopic compositions of seven of the most abundant alkylporphyrins from the Serpiano oil shale (marine, Triassic) were determined. For the C31 and C32 butanoporphyrins, values of delta 13CPDB and delta 15NAIR averaged -24.0% and -3.1%. In contrast, the C31 and C32 methylpropanoporphyrins, DPEP, and a C30 13-nor etioporphyrin had delta 13C and delta 15N values averaging -27.5 and -3.3%, respectively. Carbon and nitrogen isotopic values for kerogen averaged -30.8 and -0.9, whereas those for total extract averaged -31.6, and -4.0%. The butanoporphyrins apparently derive from a biological source different from that giving rise to the other porphyrins, their 13C enrichment not being related to carbon isotopic fractionation accompanying diagenetic reactions. The delta 15N values for all the porphyrins indicate that the depletion of 15N observed in the kerogen is of primary origin. Consistent with the very high abundance of hopanoids and methyl hopanoids in the aliphatic hydrocarbon fraction, it is suggested that cyanobacterial fixation of N2 may have been the main cause of 15N depletion.