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.

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.

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.

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.

The post-Paleozoic chronology and mechanism of 13C depletion in primary marine organic matter.

Carbon-isotopic compositions of geoporphyrins have been measured from marine sediments of Mesozoic and Cenozoic age in order to elucidate the timing and extent of depletion of 13C in marine primary producers. These results indicate that the difference in isotopic composition of coeval marine carbonates and marine primary photosynthate was approximately 5 to 7 permil greater during the Mesozoic and early Cenozoic than at present. In contrast to the isotopic record of marine primary producers, isotopic compositions of terrestrial organic materials have remained approximately constant for this same interval of time. This difference in the isotopic records of marine and terrestrial organic matter is considered in terms of the mechanisms controlling the isotopic fractionation associated with photosynthetic fixation of carbon. We show that the decreased isotopic fractionation between marine carbonates and organic matter from the Early to mid-Cenozoic may record variations in the abundance of atmospheric CO2.

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.

Compound-specific isotopic analyses: a novel tool for reconstruction of ancient biogeochemical processes.

Patterns of isotopic fractionation in biogeochemical processes are reviewed and it is suggested that isotopic fractionations will be small when substrates are large. If so, isotopic compositions of biomarkers will reflect those of their biosynthetic precursors. This prediction is tested by consideration of results of analyses of geoporphyrins and geolipids from the Greenhorn Formation (Cretaceous, Western Interior Seaway of North America) and the Messel Shale (Eocene, lacustrine, southern Germany). It is shown (i) that isotopic compositions of porphyrins that are related to a common source, but which have been altered structurally, cluster tightly and (ii) that isotopic differences between geolipids and porphyrins related to a common source are equal to those observed in modern biosynthetic products. Both of these observations are consistent with preservation of biologically controlled isotopic compositions during diagenesis. Isotopic compositions of individual compounds can thus be interpreted in terms of biogeochemical processes in ancient depositional environments. In the Cretaceous samples, isotopic compositions of n-alkanes are covariant with those of total organic carbon, while delta values for pristane and phytane are covariant with those of porphyrins. In this unit representing an open marine environment, the preserved acyclic polyisoprenoids apparently derive mainly from primary material, while the extractable, n-alkanes derive mainly from lower levels of the food chain. In the Messel Shale, isotopic compositions of individual biomarkers range from -20.9 to -73.4% vs PDB. Isotopic compositions of specific compounds can be interpreted in terms of origin from methylotrophic, chemautotrophic, and chemolithotrophic microorganisms as well as from primary producers that lived in the water column and sediments of this ancient lake.

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.

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.

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.

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.

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.

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.

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.

Association of anterior ectopic anus and partial absence of levator musculature in a woman with impaired defecation. Report of a case.

A 25-year-old nulliparous woman with adult onset constipation and slight anterior displacement of the anus underwent pelvic magnetic resonance imaging and was diagnosed with congenital hemiabsence of the levator ani sling. Impaired defecation was confirmed by anorectal function studies and defecography demonstrated an anterior rectocele, perineal descent at the upper limit of normal, and partial obstruction of defecation, which appeared related to the levator sling abnormality. To our knowledge, this combination of findings has not been previously described as a cause of adult onset constipation.

Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci.

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activated L-lactate dehydrogenase (L-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogeneous group. They exhibit a similar cell wall composition, produce predominantly D,L-lactate and have a characteristic and simple esterase pattern. Coagulase-positive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typical Staphylococcus aureus strains. They exhibit a different cell wall composition, produce only L-lactate, possess an L-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.

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.

Origin of petroporphyrins. 2. Evidence from stable carbon isotopes.

Compared with the carbon-13 isotopic composition of the ubiquitous C32DPEP (DPEP, deoxophylloerythroetioporphyrin) the heavy but equivalent carbon-13 isotopic composition for the porphyrin structures 15(2)-methyl-15,17-ethano-17-nor-H-C30DPEP and 15,17-butano-, 13,15-ethano-13(2),17-propano-, and 13(1)-methyl-13,15-ethano-13(2),17-propanoporphyrin suggests a common precursor, presumably chlorophyll c, for these petroporphyrins isolated from the marine Julia Creek oil shale and the lacustrine Condor oil shale. Similarly, the heavy but variable carbon-13 isotopic composition of 7-nor-H-C31DPEP compared with C32DPEP is consistent with an origin from both chlorophyll b and chlorophyll c3. The equivalent carbon-13 isotopic composition for 13(2)-methyl-C33DPEP compared with C32DPEP suggests a common origin resulting from a weighted average of chlorophyll inputs.

Origins of etioporphyrins in sediments: evidence from stable carbon isotopes.

In samples of the Julia Creek and Condor oil shales (Australia, Albian, and early Tertiary, respectively) etioporphyrin III is significantly depleted in 13C (4%) relative to porphyrins derived from chlorophylls. This isotopic difference suggests a large contribution from some independent source. The haem group found in cytochromes derived from microbial sources is the most likely candidate.

An isotopic study of biogeochemical relationships between carbonates and organic carbon in the Greenhorn Formation.

Carbon-isotopic compositions of total carbonate, inoceramid carbonate, micritic carbonate, secondary cements, total organic carbon, and geoporphyrins have been measured in 76 different beds within a 17-m interval of a core through the Greenhorn Formation, an interbedded limestone and calcareous shale unit of Cretaceous age (Cenomanian-Turonian) from the Western Interior Seaway of North America. Results are considered in terms of variations in the processes of primary production (which led to the biosynthesis of the molecular precursors of the geoporphyrins) and in secondary processes (those mediating the transformation of primary organic material into sedimentary total organic carbon). It is shown that the porphyrin isotopic record reflects primary isotopic variations more closely than the TOC isotopic record, and that, in these sediments, TOC is enriched in 13C relative to its primary precursor by 0.6 to 2.8%. This enrichment is attributed to isotope effects within the consumer foodweb and is associated with respiratory heterotrophy. Variations in this secondary enrichment are correlated with variations in the isotopic composition of marine carbonate. This correlation is attributed to effects of environmental changes on the marine foodweb. These may have included increased atmospheric oxygen associated with the Cenomanian-Turonian oceanic anoxic event. The isotopic fractionation associated with fixation of carbon by primary producers is observed to have varied by 1.5% during the interval of deposition. It is suggested that this change is due to a variation in the makeup of the community of primary producers and/or to a decrease in the atmospheric abundance of CO2 during the oceanic anoxic event.