Analysis of [2H7]methionine, [2H4]methionine,methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [²H₇]methionine, [²H₄]methionine, methionine, [²H₄]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [¹³C]Methionine and [¹³C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.     

Determination of [Mg2+]i – an update on the use of Mg2+-selective electrodes

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca²⁺. The development of sensors for Mg²⁺-selective electrodes has made its most striking progress only over the past few years. While the first Mg²⁺ sensor, ETH 1117, was barely able to detect physiological Mg²⁺ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca²⁺ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg²⁺-selective microelectrodes is about to start.     

Characterization of the functional domain of β2-microglobulin from the Asian seabass, Lates calcarifer

Background

β2-Microglobulin (β₂M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism.

Methodology/Principal Findings

In this study, a cDNA sequence encoding β₂M in the Asian seabass (Lates calcarifer) was identified and analyzed using in silico approaches to predict and characterize its functional domain. The β₂M cDNA contains an open reading frame (ORF) of 351 bases with a coding capacity of 116 amino acids. A large portion of the protein consists of the IG constant domain (IGc1), similar to β₂M sequences from other species studied thus far. Alignment of the IGc1 domains of β₂M from L. calcarifer and other species shows a high degree of overall conservation. Seven amino acids were found to be conserved across taxa whereas conservation between L. calcarifer and other fish species was restricted to 14 amino acids at identical conserved positions.

Conclusion/Significance

As the L. calcarifer β₂M protein analyzed in this study contains a functional domain similar to that of β₂M proteins in other species, it can be postulated that the β₂M proteins from L. calcarifer and other organisms are derived from a common ancestor and thus have a similar immune function. Interestingly, fish β₂M genes could also be classified according to the ecological habitat of the species, i.e. whether it is from a freshwater, marine or euryhaline environment.     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

COVID-19: Omicron – the latest,the least virulent,but probably not the last variant of concern of SARS-CoV-2

The Omicron variant rapidly became the dominant SARS-CoV-2 strain in South Africa and elsewhere. This review explores whether this rise was due to an increased transmission of the variant or its escape from population immunity by an extensively mutated spike protein. The mutations affected the structure of the spike protein leading to the loss of neutralization by most, but not all, therapeutic monoclonal antibodies. Omicron also shows substantial immune escape from serum antibodies in convalescent patients and vaccinees. A booster immunization increased, however, the titre and breadth of antiviral antibody response. The cellular immune response against Omicron was largely preserved explaining a satisfying protection of boosted vaccinees against severe infections. Clinicians observed less severe infection with Omicron, but other scientists warned that this must not necessarily reflect less intrinsic virulence. However, in animal experiments with mice and hamsters, Omicron infections also displayed a lesser virulence than previous VOCs and lung functions were less compromised. Cell biologists demonstrated that Omicron differs from Delta by preferring the endocytic pathway for cell entry over fusion with the plasma membrane which might explain Omicron’s distinct replication along the respiratory tract compared with Delta. Omicron represents a distinct evolutionary lineage that deviated from the mainstream of evolving SARS-CoV-2 already in mid-2020 raising questions about where it circulated before getting widespread in December 2021. The role of Omicron for the future trajectory of the COVID-19 pandemic is discussed.     

First Inactive Conformation of CK2α, the Catalytic Subunit of Protein Kinase CK2

The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2β, noncatalytic subunit of CK2). CK2α belongs to the superfamily of eukaryotic protein kinases (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix αC and in the activation segment, leading to a characteristic arrangement of catalytic key elements. For CK2α, however, no strict physiological control of activity is known. Accordingly, CK2α was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N-terminal segment on one side, and helix αC and the activation segment on the other side. We report here the structure of a C-terminally truncated variant of human CK2α in which the enzyme adopts a decidedly inactive conformation for the first time. In this CK2α structure, those regulatory key regions still are in their active positions. Yet the glycine-rich ATP-binding loop, which is normally part of the canonical anti-parallel β-sheet, has collapsed into the ATP-binding site so that ATP is excluded from binding; specifically, the side chain of Arg47 occupies the ribose region of the ATP site and Tyr50, the space required by the triphospho moiety. We discuss some factors that may support or disfavor this inactive conformation, among them coordination of small molecules at a remote cavity at the CK2α/CK2β interaction region and binding of a CK2β dimer. The latter stabilizes the glycine-rich loop in the extended active conformation known from the majority of CK2α structures. Thus, the novel inactive conformation for the first time provides a structural basis for the stimulatory impact of CK2β on CK2α.     

Conformational Change of the Rieske [2Fe–2S] Protein inCytochrome bc 1 Complex

Structures of mitochondrial bc ₁ complex have been reported based on four different crystalforms by three different groups. In these structures, the extrinsic domain of the Rieske [2Fe–2S]protein, surprisingly, appeared at three different positions: the c ₁ position, where the [2Fe–2S]cluster exists in close proximity to the heme c ₁; the b position, where the [2Fe–2S] clusterexist in close proximity to the cytochrome b; and the intermediate position where the[2Fe–2S] cluster exists in between c ₁ and b positions. The conformational changes betweenthese three positions can be explained by a combination of two rotations; (1) a rotation of theentire extrinsic domain and (2) a relative rotation between the cluster-binding fold and thebase fold within the extrinsic domain. The hydroquinone oxidation and the electron bifurcationmechanism at the Q_P binding pocket of the bc ₁ complex is well explained using theseconformational changes of the Rieske [2Fe–2S] protein.     

The biogenesis of [Fe–S] clusters: the role of the unorthodox ABC ATPase,SufC, and the wider implications for understanding iron metabolism

Abstract

Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H₂O₂, F₂-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.     

Does the use of DM-nitrophen,nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence?

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr-5, and diazo-2 were studied alone, and in the presence of indo-1, to investigate potential interactions that would make the simultaneous manipulation and ratiometric measurement of the intracellular Ca2+ concentration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo-2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen, nitr-5 itself was found to be quite fluorescent, and this fluorescence was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of all the investigated compounds were used to define experimental conditions and calibration procedures that make possible simultaneous measurement and manipulation of the intracellular Ca2+ concentration.     

The mechanism of antimalarial action of the ruthenium(II)–chloroquine complex [RuCl2(CQ)]2

The mechanism of antimalarial action of the ruthenium-chloroquine complex [RuCl(2)(CQ)](2) (1), previously shown by us to be active in vitro against CQ-resistant strains of Plasmodium falciparum and in vivo against P. berghei, has been investigated. The complex is rapidly hydrolyzed in aqueous solution to [RuCl(OH(2))(3)(CQ)](2)[Cl](2), which is probably the active species. This compound binds to hematin in solution and inhibits aggregation to beta-hematin at pH approximately 5 to a slightly lower extent than chloroquine diphosphate; more importantly, the heme aggregation inhibition activity of complex 1 is significantly higher than that of CQ when measured at the interface of n-octanol-aqueous acetate buffer mixtures under acidic conditions modeling the food vacuole of the parasite. Partition coefficient measurements confirmed that complex 1 is considerably more lipophilic than CQ in n-octanol-water mixtures at pH approximately 5. This suggests that the principal target of complex 1 is the heme aggregation process, which has recently been reported to be fast and spontaneous at or near water-lipid interfaces. The enhanced antimalarial activity of complex 1 is thus probably due to a higher effective concentration of the drug at or near the interface compared with that of CQ, which accumulates strongly in the aqueous regions of the vacuole under those conditions. Furthermore, the activity of complex 1 against CQ-resistant strains of P. falciparum is probably related to its greater lipophilicity, in line with previous reports indicating a lowered ability of the mutated transmembrane transporter PfCRT to promote the efflux of highly lipophilic drugs. The metal complex also interacts with DNA by intercalation, to a comparable extent and in a similar manner to uncomplexed CQ and therefore DNA binding does not appear to be an important part of the mechanism of antimalarial action in this case.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Concerning the presence of the cytokinin,N 6-(Δ2-isopentnyl) adenine,in cultures of Corynebacterium fascians

Summary Corynebacterium fascians causes a fasciation disease in a number of dicotyledons and this disease appears to be caused by compounds with cytokinin activity elaborated by the infecting bacteria. Extractions of C. fascians in late logarithmic phase under conditions where the pH never falls below 7.0 yield about 2 g/l of N ⁶-(², a potent cytokinin. If a mild acidification step is included in the extraction procedure the yield increases to about 12 g/l. This is due to release of N ⁶-(²-isopentenyl)adenine from C. fascians tRNA during the extraction procedure. In terms of total cytokinin activity present in C. fascians cultures, N ⁶-(²-isopentenyl)adenine appears to be a minor component.     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

The Synthesis,Structure, and Conformation of 2′-Deoxy Analogues of „Fat“ Xanthine Nucleosides,Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The syntheses of 1-(2-deoxy-β-D-erythro-pentofuranosyl)-4,5,7,8-tetrahydro-6H-imidazo[4,5-e] [1,4]diazepine-5,8-dione (9β), its 3-glycosyl regioisomer (16β), and their respective α anomers (9α and 16α), are reported. Conformational and configurational studies, employing ¹H NMR NOE and CD spectroscopy, are described. The single-crystal X-ray structural analysis of 9β is presented. The attempted enzymic glycosylation of the heterocyclic base 6 with a bacterial purine nucleoside phosphorylase was not successful.