Dinoflagellate cyst biostratigraphy of the Coniacian–Santonian (Upper Cretaceous): New data from the English Chalk

Results are presented here for the first chalk-based, high-resolution quantitative study of organic-walled dinoflagellate cysts (dinocysts) from the entire Coniacian–Santonian Chalk successions in east Kent and on the Isle of Wight. The lithostratigraphy and dinocyst records (137 taxa) of seven sections are presented, and the stratigraphic ranges of taxa are constrained relative to stage and zonal boundaries, located using extensive macrofossil data. Results are integrated with a previous complementary study of the Isle of Wight Santonian to test and refine existing dinocyst bioevent schemes. Sixteen dinocyst events are proposed as a sequence of biostratigraphic datum levels for the lower Coniacian to uppermost Santonian which, based on average sedimentation rates, represent an average temporal resolution of around 360 kyr. The event stratigraphy forms a basis for the first high-resolution correlation study of quantitative dinocyst data from the Upper Cretaceous of NW Europe. A new genus Culversphaera Prince, Jarvis, Pearce et Tocher gen. nov. is proposed with the new combination Culversphaera velata Prince, Jarvis, Pearce et Tocher gen. et comb. nov. Five new species: Ellipsodinium membraniferum Prince, Jarvis, Pearce et Tocher sp. nov., Senoniasphaera macroreticulata Prince, Jarvis, Pearce et Tocher sp. nov., Senoniasphaera whitenessii Prince, Jarvis, Pearce et Tocher sp. nov., Xenascus spinatus Prince, Jarvis, Pearce et Tocher sp. nov. and X. yunii sp. nov., and the new subspecies S. protrusa congrensa Prince, Jarvis, Pearce et Tocher subsp. nov. are described.     

The Cenomanian–Turonian boundary event, OAE2 and palaeoenvironmental change in epicontinental seas: New insights from the dinocyst and geochemical records

Organic-walled dinoflagellate cyst (dinocyst) and geochemical records across the Cenomanian–Turonian boundary (CTB) are presented for the NW European reference section at Eastbourne, England. Dinocyst and nannofossil fertility indexes indicate that an upwelling-driven productivity pulse accompanied a eustatic sea-level fall that preceded, by at least 40 kyr, the global rise in δ¹³C values that marks the onset of Oceanic Anoxic Event 2 (OAE2) and the deposition of black shales in the deep ocean. A marine productivity collapse in the latest Cenomanian is evidenced by the falling absolute and relative abundance of peridinioid dinocysts, believed to be the product of heterotrophic dinoflagellates. This biotic change accompanied transgression and sharply rising sea-surface temperatures, following an Atlantic-wide episode of short-lived cooling. Geochemical tracers provide evidence of Caribbean–Colombian plateau volcanism. The increase in water depth caused by the latest Cenomanian transgression resulted in less eutrophic waters in epicontinental seas, where CTB biotic turnover was driven largely by water-mass changes rather than oxygen depletion. The species richness/absolute abundance ratio of dinocysts is proposed as a water-mass stability proxy.     

Thermal Preference of Juvenile Dover Sole (Solea solea) in Relation to Thermal Acclimation and Optimal Growth Temperature

Dover sole (Solea solea) is an obligate ectotherm with a natural thermal habitat ranging from approximately 5 to 27°C. Thermal optima for growth lie in the range of 20 to 25°C. More precise information on thermal optima for growth is needed for cost-effective Dover sole aquaculture. The main objective of this study was to determine the optimal growth temperature of juvenile Dover sole (Solea solea) and in addition to test the hypothesis that the final preferendum equals the optimal growth temperature. Temperature preference was measured in a circular preference chamber for Dover sole acclimated to 18, 22 and 28°C. Optimal growth temperature was measured by rearing Dover sole at 19, 22, 25 and 28°C. The optimal growth temperature resulting from this growth experiment was 22.7°C for Dover sole with a size between 30 to 50 g. The temperature preferred by juvenile Dover sole increases with acclimation temperature and exceeds the optimal temperature for growth. A final preferendum could not be detected. Although a confounding effect of behavioural fever on temperature preference could not be entirely excluded, thermal preference and thermal optima for physiological processes seem to be unrelated in Dover sole.     

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg⁻¹ protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min⁻¹·mg⁻¹ protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.     

Tectonically controlled late cretaceous terrestrial to neritic deposition (Northern Calcareous Alps, Tyrol, Austria)

Summary The Turonian to Santonian terrestrial to neritic succession (Lower Gosau Subgroup) in the Northern Calcareous Alps of the eastern part of the Tyrol, Austria, provides an example for deposition on a compartmentalized, narrow, microtidal to low-mesotidal, wave-dominated, mixed siliciclastic-carbonate shelf. The shelf was situated in front of a mainland with a relatively high, articulated relief, and underwent distinct changes in facies architecture mainly as a result of tectonism. The investigated succession was deposited above a deeply incised, articulated truncation surface that formed when the Eo-Alpine orogen, including the area of the future Northern Calcareous Alps, was uplifted and subaerially eroded. Distinct facies associations were deposited from (1) alluvial fans and fan deltas, (2) rivers, (3) siliciclastic lagoonal to freshwater marsh environments, (4) areally/temporally limited carbonate lagoons, (5) transgressive shores, (6) siliciclastic shelf environments, and (7) an aggrading carbonate shelf. During the Turonian to Coniacian, the combination of high rates of both subsidence and sediment accumulation, and a narrow shelf that was compartmentalized with respect to (a) morphology of the substratum, (b) fluviatile input of siliciclastics and contemporaneous input of carbonate clasts from fan deltas, (c) deposition of shallow-water carbonates, and (d) water energy and-depth gave rise to an exceptionally wide spectrum of facies as a distinguishing feature of the succession. With the exception of facies association 7, which formed only once, depositional sequences in the Turonian to Coniacian interval contain all of the facies associations 1 to 6. During Turonian to Coniacian times, the shelf was microtidal to low-mesotidal, and was dominated by waves, storm waves and storm-induced currents. In vegetated marshes, schizohaline to freshwater marl lakes existed. Transgressions occurred onto fan deltas and in association with estuaries, or in association with gravelly to rocky shores. The transgressive successions, including successions deposited from transgressive rocky carbonate shores, are overlain by regressive successions of shelf carbonates or shelf siliciclastics. Deposition of shallow-water carbonates generally occurred within lagoons and over short intervals of time. A „catch-up” succession of shelf carbonates about 100 m thick accumulated only in an area protected from siliciclastic input. In its preserved parts, the Turonian to Coniacian succession does not record deposition adjacent to major active faults. Lateral changes in thickness result mainly from onlap onto the articulated basal truncation surface. Subsidence most probably was controlled by major detachment faults outside the outcrop area, and/or was distributed over a wide area in association with secondary faults above the major detachments. During Coniacian to Early Santonian times, both the older substratum and the overlying Turonian-Coniacian succession were subaerially exposed, faulted and deeply eroded. The following Early Santonian transgression ensued with rocky carbonate shores ahead of a sandy, narrow shoreface-inner shelf environment and a deeper shelf with intermittentlydysaerobic mud. The transgression was associated with the influx of cooler and/or nutrient-rich waters, and heralds an overall deepening. Still during the Early Santonian, the deepening was interrupted by another phase of subaerial exposure. Subsequently, a short phase of shelf deposition was terminated by deepening into bathyal depths.     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

Late Cretaceous radiolarian biochronology of the Pedro Brand section,Tireo Group,eastern Central Cordillera,Dominican Republic: A contribution to the stratigraphy of the Caribbean Large Igneous Province

The Tireo Group in the eastern Central Cordillera (Dominican Republic) is part of the Jarabacoa Block, composed of a Pacific-type Jurassic ocean floor (Loma La Monja, overlain by the El Aguacate ribbon Chert), intruded and overlain by an early CLIP (Carribean Large Igneous Province)-type plateau, the Duarte Complex, which is in turn unconformably overlain by arc-type rocks of the Tireo Group. This group exhibits a 3-km thick sequence of arc-related volcanic and volcano-sedimentary rocks, including tuffaceous chert and mudstone studied for radiolarians in this paper. The Siete Cabezas Formation, considered to be the last Campanian–Maastrichtian CLIP-type volcano-sedimentary sequence, overlies the Tireo Group. A controversy about earlier radiolarian dating of the Tireo Group, considered until now as part of the Siete Cabezas Formation, encouraged us to study a well-exposed section located 3 km NE of Pedro Brand village. Seven samples of laminated siliceous mudstones and cherts yielded around 40 common and well-preserved radiolarian taxa. Based on maximum ranges of taxa published in several regional zonations and on a comparison with a Turonian–Coniacian sample calibrated by planktonic foraminifera (the Deva Beds from Romania), we determine a Turonian–Coniacian age for the Pedro Brand section. A ⁴⁰Ar–³⁹Ar whole rock age of 75.1 ± 1.1 Ma, obtained in a basalt dyke crosscutting the radiolarian bearing rocks, provides a Late Campanian consistent minimum age for the pelagic–hemipelagic Pedro Brand section. Including the re-interpretation of earlier radiolarian work, we conclude that the studied rocks of the Tireo Group are older than the Maastrichtian ⁴⁰Ar–³⁹Ar ages on plagioclase of the Siete Cabezas Formation. The studied dyke in the Pedro Brand section geochemically resembles the overlying Siete Cabezas and Pico Duarte basalts and could be a feeder dyke of those. However, a tectonic superposition of the Siete Cabezas cannot be excluded, since earlier ⁴⁰K–⁴⁰Ar basalt ages of this unit are Aptian–Albian and Cenomanian–Turonian. The Jarabacoa Block is considered as the most complete outcrop section of Pacific ocean crust overlain by a first (Aptian–Albian) phase of CLIP-type activity, followed by the development of a Cenomanian–Santonian intraoceanic arc, which is in turn overlain by a late Campanian–Maastrichtian CLIP-like phase.     

Hemodynamic Changes in Left Anterior Descending Coronary Artery and Anterior Interventricular Vein during Right Ventricular Apical Pacing: A Doppler Ultrasound Study in Open Chest Beagles

Objective

The aim of this study was to quantify the effects of right ventricular apical pacing (RVAP) on hemodynamics in left anterior descending coronary artery (LAD) and anterior interventricular vein (AIV) contrast to baseline condition in open chest beagles using Doppler ultrasound imaging.

Methods

In 6 anesthetized open chest beagles, the spectral Doppler waveforms of the middle segmental LAD and the AIV were acquired with a 5 MHz linear array transducer at baseline condition and during RVAP. The aortic pressure-time curves were recorded synchronously. The Doppler hemodynamic parameters of the LAD and AIV at both states were derived and compared.

Results

The spectral Doppler waveforms of the LAD had a principal diastolic positive wave (Dp), which heelled by a momentary negative wave and a positive wave during early systole at baseline condition. During RVAP, an additional negative wave appeared in the LAD at late systole. The duration of the Dp shortened (227.83±12.16 ms vs 188.50±8.97 ms, P<0.001), and the acceleration of the Dp decreased (11.85±2.22 m/s² vs 3.54±0.42 m/s², P<0.001). The spectral Doppler waveforms of the AIV only had a principal positive wave (Sp) at baseline condition, but an additional diastolic negative wave appeared during RVAP. The duration of the Sp shortened (242.99±7.98 ms vs 215.38±15.44 ms, P<0.001), and the acceleration of the Sp decreased (9.61±1.93 m/s² vs 1.01±0.11 m/s², P<0.001).

Conclusions

Obvious hemodynamic changes in the LAD and AIV during RVAP were observed, and these abnormal flow patterns in epicardial coronary arteries and vena coronaria may be sensitive and important hints of the disturbed cardiac electrical and mechanical activity sequences.     

Late Albian dinoflagellate cyst paleobiogeography as indicator of asymmetric sea surface temperature gradient on both hemispheres with southern high latitudes warmer than northern ones

Late Albian temperature sensitive dinoflagellate cysts are characterized in order to better understand mid-Cretaceous sea surface temperature gradients. Distribution maps of thirty-seven species recovered from one hundred fifty outcrops and deep sea drilling holes (ODP, DSDP) Sites located from low and high paleolatitudes over the two hemispheres (75°N–70°S) are encountered. Fifty years of published data available in eighty-seven articles have been considered and synthesized using a database coupled with a Geographical Information System (GIS). The continuous and disjoint biogeographic patterns of dinocyst species along latitudes define seven climatic belts, four in the northern hemisphere (high, mid-high, mid-low, low latitude belts) and three in the southern hemisphere (low, mid-low, mid-high latitude belts). Dinocysts restricted along latitudes are temperature sensitive species. Limit ranges of temperature sensitive dinocysts of mid-low and mid-high latitude belts reveal mixing belts, located at 40–45°N and 50°–70°S. They represent major palaeofrontal systems as paleosubtropical fronts with strongly mixed water column. The northern frontal system (40–45°N) was located as in the modern ocean. The large southern frontal system (50°S–70°S) was 10°–20° poleward to Antarctica. Semi-quantitative temperature range limits for mid-Cretaceous dinoflagellates and SST gradients in the two hemispheres are suggested by setting dinocyst climatic belts against estimated δ¹⁸O temperature curve from fish teeth. A paleoecological classification is suggested. Latitudinal distribution of extant temperature sensitive dinoflagellate cysts follows the asymmetric modern temperature gradient. Asymmetric latitudinal ranges of Late Albian dinocyst species of mid-low latitude belts restricted between 45°N and 70°S demonstrate asymmetric temperature gradients with southern high latitudes being warmer than northern high latitudes.     

Bifunctional Homodimeric Triokinase/FMN Cyclase: CONTRIBUTION OF PROTEIN DOMAINS TO THE ACTIVITIES OF THE HUMAN ENZYME AND MOLECULAR DYNAMICS SIMULATION OF DOMAIN MOVEMENTS*

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and k_cat and K_m were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr¹¹² (hydrogen bonding of ATP adenine to K in the closed active center), His²²¹ (covalent anchoring of dihydroxyacetone to K), Asp⁴⁰¹ and Asp⁴⁰³ (metal coordination to L), and Asp⁵⁵⁶ (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His²²¹ point mutant acted specifically as a cyclase without kinase activity.     

Atypical Biosynthetic Properties of a Δ12/ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Δ¹²-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C₁₈ substrates with ν+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (ν). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30°C versus 15°C). Thus, while maintaining the hallmark activity of the fatty acyl Δ¹²-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.     

First record of the Late Cretaceous starfish Metopaster parkinsoni (Forbes, 1848) in Iran

A body fossil of the starfish Metopaster parkinsoni was collected from chalky rocks of the Coniacian – Santonian transition in the Abderaz Formation at the Padeha section, east of Mashhad (Kopet-Dagh, Iran). The species is common in the English Chalk and is well known from the Cenomanian to the Maastrichtian of Europe. The new Iranian occurrence of M. parkinsoni extends its geographical range of 700 km to the South and 2500 km to the East. The species proliferated in upper offshore environments of the North Tethyan continental shelves. The outstanding occurrence of many well-preserved specimens during the Coniacian – Santonian transition in Europe and Iran may be related to favorable taphonomic conditions driven by the Ocean Anoxic Event (OAE III).     

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

3-Ketosteroid Δ¹-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ¹-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ¹-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr¹¹⁹, Tyr³¹⁸, Tyr⁴⁸⁷, and Gly⁴⁹¹. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr³¹⁸, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr⁴⁸⁷ and the backbone amide of Gly⁴⁹¹. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr⁴⁸⁷ and Gly⁴⁹¹ work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr¹¹⁹, the general base Tyr³¹⁸ abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.     

Calcareous dinoflagellate cyst distributions in surface sediments from upwelling areas off NW Africa, and their relationships with environmental parameters of the upper water column

Only very few studies have focussed on the spatial distribution and ecology of calcareous dinoflagellate cysts (dinocysts) in upwelling areas. Here, distributions of individual calcareous dinocyst species in surface sediment samples from the coastal upwelling zone off NW Africa and their relationships with known environmental parameters of the (sub-)surface waters have been analysed in order to enhance our knowledge on their modern distribution patterns and to determine the ecological significance and palaeoenvironmental reconstruction potential of each calcareous dinocyst species within this exemplary high-productivity region. In addition to calculating relative cyst abundances, well-constrained sedimentation rates have allowed the calculation of dinocyst accumulation rates (cysts cm^− 2 ka^− 1) for most of the surface sample positions, thus providing a much more accurate estimation of actual calcareous dinocyst production in the upper water column than could be warranted in similar studies so far. Distinct differences in the geographic distribution patterns of nine species were observed. In general, high accumulation rates of calcareous dinocysts were found around and south of 29°N and offshore the westernmost Canary Island La Palma, with slightly lower accumulation rates in the upwelling-influenced areas off Cape Yubi and in the upwelling filament area off Cape Ghir. Multivariate ordination techniques were applied in order to compare the cyst accumulation rates of individual species with physical parameters (temperature, salinity, density, mixed layer depth) and the trophic state (nitrate, phosphate and chlorophyll concentrations) of the overlying (sub-)surface waters. All species were found to relate significantly to one or more of the environmental parameters, partly confirming previous results on cyst ecology but also providing new information which will be useful for future palaeoenvironmental reconstructions within upwelling areas.     

Di(1,N-ethenoadenosine)5′, 5-P,P-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N⁶-ethenoadenosine) 5′, 5-P¹, P⁴-tetraphosphate, ε-(Ap₄A), a fluorescent analog of Ap₄A has been synthesized by reaction of 2-chloroacetaldehyde with Ap₄A. At neutral pH this Ap₄A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λ_exc 307 nm, λ_em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ε(Ap₄A) and catabolize the resulting ε-nucleotide moieties up to ε-Ado.     

Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β₂ subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr³⁹⁴ of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K_m values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K_ic (competitive inhibition) value of 18 ± 9 μM and a K_iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Desulfotomaculum and Methanobacterium spp. Dominate a 4- to 5-Kilometer-Deep Fault

Alkaline, sulfidic, 54 to 60°C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO₄²⁻ in the presence of high pH, high concentrations of H₂ and CH₄, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO₄²⁻ reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.     

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H^N, ¹⁵N, ¹³C^α and ¹³C^β atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in ²H, ¹³C and ¹⁵N. The secondary structure determined on the basis of observed ¹³C^α secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein–protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intrasubunit ilvBα/ilvBβ domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.     

Geochemical Evidence of the Seasonality,Affinity and Pigmenation of Solenopora jurassica

Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ¹³C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ¹⁸O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ¹⁸O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ¹³C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil algae.