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PubMed2006

Modulation of the axial water hydrogen-bonding properties by chemical modification of the substrate in resting state, substrate-bound heme oxygenase from Neisseria meningitidis; coupling to the distal H-bond network via ordered water molecules.

The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitidis has been systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of 1H NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisotropy increasing in the order of substituent formyl < vinyl < methyl. The magnetic anisotropy is axial and oriented by the axial Fe-His23 bond, and while individual anisotropies have uncertainties of approximately 5%, the relative values of deltachi (and the zero-field splitting constant, D proportional, variant deltachi(ax)) are defined to 1%. The unique changes in the axial field strength implied by the variable zero-field splitting are in accord with expectations for the axial water serving as a stronger H-bond donor in the order of hemin substituents formyl > vinyl > methyl. These results establish the axial anisotropy (and D) as a sensitive probe of the H-bonding properties of a ligated water in resting-state, substrate complexes of heme oxygenase. Correction of observed labile proton chemical shifts for paramagnetic influences indicates that Gln49 and His53, some approximately 10 angstroms from the iron, sense the change in the ligated water H-bonding to the three nonligated ordered water molecules that link the two side chains to the iron ligand. The present results augur well for detecting and characterizing changes in distal water H-bonding upon mutagenesis of residues in the distal network of ordered water molecules and strong H-bonds.

Anisotropy↗

PubMed1977

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.

Alanine↗

PubMed1995

Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase.

1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Brattleboro rats caused significant reductions in myocardial ATP, creatine, and total creatine concentrations and an accumulation of tissue lactate when compared with control animals. Administration of creatine (0.63 mg ml-1 day-1 in the drinking water) for 4 weeks elevated myocardial creatine and total creatine concentrations and reduced lactate accumulation, but did not significantly affect ATP or phosphocreatine (PCr). Concurrent treatment with creatine and L-NAME prevented the reduction in creatine and total creatine concentrations, and significantly attenuated the accumulation of lactate and the reduction in ATP seen with L-NAME alone. 3. In a second series of experiments, 4 weeks treatment with L-NAME and creatine plus L-NAME increased mean arterial blood pressure in conscious Brattleboro rats. Hearts isolated from these animals showed decreased coronary flow and left ventricular developed pressure (LVDP), and total mechanical performance. Treatment with creatine alone had no measurable effect on either mean arterial blood pressure or coronary flow in isolated hearts. However, there was an increase in LVDP, but not in total mechanical performance, because there was a bradycardia. 4. These results indicate that creatine supplementation can attenuate the metabolic stress associated with L-NAME administration and that this effect occurs as a consequence of the action of creatine on myocardial energy metabolism.

Adenosine Triphosphate↗

PubMed1997

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

Calcium Channel Blockers↗

PubMed2004

Post-synaptic N-methyl-d-aspartate signalling in hippocampal neurons of rat: spillover increases the impact of each spike in a short burst discharge.

High-frequency burst discharges in hippocampus typically consist of less than ten spikes fired at frequencies too high to be followed by a post-synaptic neuron. How significant are these numbers for synaptic signalling? We have measured the N-methyl-d-aspartate (NMDA) component of the excitatory post-synaptic current (EPSC(NMDA)) in hippocampal CA1 neurons of rat after burst discharge of variable duration. The synaptic facilitation is accompanied by a slow-down of the EPSC(NMDA) which develops on a spike-to-spike basis. Consequently the charge transferred by the after-burst EPSC(NMDA) is increased with each spike. The phenomenon is most probably due to the spillover-mediated recruitment of extrasynaptic NMDA receptors. In terms of post-synaptic signalling it dramatically increases the impact of each spike in a short burst discharge.

Action Potentials↗

PubMed1995

Discrimination in resolving systems. II: Ephedrine-substituted mandelic acids.

Binary diastereomeric (-) (1R,2S)-ephedrine salts of various mandelic acids obtained from 95% ethanol show considerable differences in solubility. Structures and some properties of the less-soluble (L) and more-soluble (M) solid phases of (-)-ephedrine with unsubstituted mandelic acid, 2-, 3-, and 4-monosubstituted halo (F, Cl, Br) mandelic acids, and 3- and 4-methylmandelic acids have been determined. Salts were found to be binary, without solvent of crystallization, and composed of double-layered arrays of alternating anions and cations linked by H-bonds normal to the layers. H-bonding links charged donors and acceptors usually along a crystallographic 2-fold screw axis. A striking discrimination is evident in that the (2R)-mandelate salts typically display a compact four-atom chain as the H-bonding repeating unit [+N--H...O(-C(-)--O)...H-N', C2(1)(4)] while the (2S)-mandelate salts adopt a more dimensionally variable six-atom chain repeating unit [+N--H...O--C(-)--O...H--N', C2(2)(6)]. Two distinct packing schemes display the shorter H-bonding chain of the (2R)-mandelates which always occurs with ephedrinium ions in the fully extended conformation. Slightly greater packing efficiency and H-bonding energies of the (2R)-mandelate salts correlates with increased fusion points, lower solubilities (95% ethanol), and higher heats of fusion relative to the phase adopted by their diastereoisomers. In contrast, (2S)-mandelate salts exhibit considerably more structural variability involving all three major ephedrinium conformations, and at least four distinct packing motifs. Mandelates with larger 3'-substituents (Cl, Br, methyl) show similar property discriminations, but these occur with an opposing trend, that is, between phases in which the less-soluble salts contain (2S)-mandelates. Salts with 2-bromomandelate do not show property disparities and their structures are dissimilar to the other phases.

Chemical Phenomena↗

PubMed1996

Multivariate analysis of capillary electrophoresis separation conditions for Z-E isomers of clomiphene.

Plackett-Burman (P-B) experimental design has been used to optimize the factors affecting separation of Z and E isomers of clomiphene (zuclomiphene and enclomiphene respectively) using capillary electrophoresis. The P-B design was used to simultaneously investigate the following five factors: buffer ionic strength, buffer pH, heptakis (2,3,6-tri-o-methyl) beta-cyclodextrin (TMCD) concentration, methanol concentration and injection time, each at three levels. In addition to these, a dummy variable was added to estimate the variability of the system. Effects on resolution and analysis time were calculated. Based on the information gained from the P-B design, the following set of conditions was chosen: 100 mM phosphate buffer pH 2.3, 5 mM TMCD, 5% methanol, and 1.7 s hydrodynamic injection time. These conditions gave well-resolved peaks for zuclomiphene and enclomiphene.

Clomiphene↗

PubMed2006

Development of a harmonised method for the profiling of amphetamines: IV. Optimisation of sample preparation.

The suitability of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for the preparation of impurity extracts intended for gas chromatographic profiling analyses of amphetamine were evaluated. Both techniques were optimised with respect to the extraction of selected target compounds by use of full factorial designs in which the variables affecting the performance were evaluated. Test samples consisted of amphetamine synthesised by the Leuckart reaction, by reductive amination of benzyl methyl ketone and by the nitrostyrene route. The performance of LLE and SPE were comparable in terms of repeatability and recovery of the target compounds. LLE was considered the better choice for the present harmonised amphetamine profiling method due to the lack of information on the long-term stability of SPE columns.

Journal Article↗

PubMed2001

Impacts of pulsatile systemic circulation on endothelium-derived nitric oxide release in anesthetized dogs.

BACKGROUND: The effects of pulsatile flow on endothelium-derived nitric oxide-mediated vasodilation are not fully elucidated in an in vivo model. METHODS: A left ventricular assist device was established in 10 anesthetized dogs with a centrifugal pump and an air-driven pneumatic pump. The systemic circulation was subjected to step changes in the frequency of pulse (0, 30, 60, and 120 bpm with a fixed pulse pressure of 50 mm Hg), and in the amplitude of pulse (0, 20, and 50 mm Hg with a fixed pulse rate of 120 bpm). Hemodynamic variables and calculated total systemic vascular resistance were compared before and after the administration of N(G)-Nitro-L-arginine Methyl Ester (L-NAME) (20 mg/kg). Plasma NO2-/NO3- concentration levels were also measured. RESULTS: Total systemic vascular resistance significantly decreased while plasma NO2-/No3- concentration increased in response to the rise in both pulse rate and pulse pressure. However, L-NAME significantly diminished these effects of pulsatile flow. CONCLUSIONS: Both the frequency and the amplitude of pulse wave in the systemic circulation are significant independent stimuli for endothelium-derived nitric oxide-mediated vasodilation in vivo.

Animals↗

PubMed2002

Total and methylmercury residues in cartilaginous fish from Mediterranean Sea.

Concentrations of mercury and methylmercury residues were determined in the muscle tissue of three cartilaginous fishes Chimaera monstrosa (ghostshark), Torpedo nobiliana (electric ray) and Myliobatis aquila (eagle ray) from the Mediterranean Sea. The highest mean levels of total mercury were detected in ghostshark (3.14 mg/kg wet weight), followed by electric ray (2.42 mg/kg wet weight), and eagle ray (0.83 mg/kg wet weight). Such a variability is influenced by quite a number of factors, among which size, fish ecology and feeding habits. The percentages of the methylated form to total mercury ranged from a minimum of 72% in eagle ray to a maximum of 83% in ghostshark. Relationship between specimen size and mercury and methylmercury concentrations were found in ghostshark species.

Animals↗

PubMed2001

Estimating temperature dependence of solubility and octanol-water partition coefficient for organic compounds using RP-HPLC.

Temperature dependence data for physical-chemical properties is increasingly required for modelling the fate of chemicals in the environment. Solubility and octanol-water partition coefficient (Kow) are among the most important parameters. A simple and fast method is presented to determine solubility and Kow of organic chemicals at different temperatures (5 degrees C, 15 degrees C, 25 degrees C, 35 degrees C) utilising a variable temperature RP-HPLC column. Correlations between capacity factors (k') and solubility and Kow were determined for some halogenated and methylated benzenes and showed that this approach could be used to predict acceptable results. New values for solubility and Kow as function of temperature for several compounds are presented.

Chlorobenzenes↗

PubMed2000

At-line monitoring of a submerged filamentous bacterial cultivation using near-infrared spectroscopy.

The use of near infra-red spectroscopy (NIRS) to monitor a submerged filamentous bacterial bioprocess was investigated. An industrial strain of the filamentous bacterium Streptomyces fradiae was cultured in a 12 litre stirred tank reactor (STR) using a complex medium. This mycelial 4 phase (oil, water, gas and solid) system produced highly complex and variable matrices, therefore monitoring such a complex fluid with NIRS represented a considerable challenge. Nevertheless, successful models for four key analytes (methyl oleate, glucose, glutamate and ammonium) were built at-line (rapid off-line) using NIRS. In the present study, the methods used to formulate, select and validate the models for the key analytes are discussed, with particular emphasis on how the model performance can be critically evaluated. Since previous reports on NIRS in monitoring bioprocesses have either involved simpler matrices, or, in filamentous systems, have not discussed how NIRS models can be critically assessed, the emphasis in the present study on providing an insight into the modelling process in such a complex matrix, may be particularly important to the applicability of NIRS to such industrial bioprocesses.

Journal Article↗

PubMed2001

A simplified one-pot synthesis of 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy.

9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG, 2) has been synthesized by nucleophilic substitution of N(2)-(p-anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethoxy)-3-toluenesulfonyloxy-2-propoxy]methyl]guanine (1) with potassium [18F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 degrees C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 degrees C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 degrees C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 degrees C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126 +/- 0.022, n=5 and 0.165 +/- 0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ( approximately 5-30 microg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains approximately 10-25 microg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is readily amenable for automation.

Chemical Phenomena↗

PubMed1998

Synthesis and Conformational Characteristics of Inherently Chiral Monoalkyl Ethers of p-tert-Butyldihomooxacalix[4]arene.

The synthesis of four inherently chiral monoalkyl ethers (methyl, ethyl, allyl, and benzyl), derived from p-tert-butyldihomooxacalix[4]arene, is described. Their conformational features were studied by variable temperature (1)H NMR spectroscopy in solvents with different polarity, such as chloroform (or CDCl(2)CDCl(2)), acetone, DMSO, and pyridine. Coalescence temperatures and DeltaG() were determined in CDCl(2)CDCl(2) and pyridine solutions. Monomethyl ether has a T(c) of 86 degrees C in CDCl(2)CDCl(2) and of -8 degrees C in pyridine, and the other derivatives are conformationally immobilized (DeltaG() >> 20 kcal mol(-1) in both solvents). The cone conformation, obtained for all monoethers, was confirmed by (13)C and NOESY spectra and also from a series of NOE 1D experiments. Complete assignment of both proton and carbon NMR spectra was achieved for the monomethyl ether by a combination of COSY, HMQC, and selective INEPT experiments, in chloroform at room temperature. Inherent chirality for all compounds was demonstrated by the addition of Pirkle's reagent to CDCl(3) solutions of monoethers derivatives, causing duplication of the NMR proton signals.

Journal Article↗

PubMed1979

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

Animals↗

PubMed2005

Halalkalicoccus tibetensis gen. nov., sp. nov., representing a novel genus of haloalkaliphilic archaea.

A haloalkaliphilic archaeon (strain DS12T) isolated from Lake Zabuye, the Tibetan Plateau, China, was characterized to elucidate its taxonomy. The strain was aerobic, chemo-organotrophic, and grew optimally at 40 degrees C, pH 9.5-10.0 and 3.4 M NaCl. Cells of strain DS12T were non-motile cocci and stained Gram-variable. The major polar lipids of strain DS12T were diphytanyl and phytanyl-sesterterpanyl diether derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Phylogenetic analysis revealed that the strain formed a distinct lineage within the family Halobacteriaceae. The low 16S rRNA gene sequence similarity values to its closest relatives (91.5-92.5 %) and its signature bases both suggest that the strain has no close affinity with any members of the family Halobacteriaceae with validly published names. Therefore, it is proposed that strain DS12T (=AS 1.3240T=JCM 11890T) represents the type strain of a novel species in a new genus, Halalkalicoccus tibetensis gen. nov., sp. nov.

Base Sequence↗

PubMed2000

Bovine beta-lactoglobulin: interaction studies with palmitic acid.

Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

Animals↗

PubMed2025

Self-regulating gene therapy ameliorates phenotypes and overcomes gene dosage sensitivity in a mouse model of Rett syndrome.

Conventional methods of gene transfer lead to inconsistent transgene expression within cells. This variability can be problematic, particularly in conditions like Rett syndrome (RTT), a neurological disorder caused by mutations in the MECP2 (methyl-CpG binding protein 2) gene, because overexpression of MECP2 can also cause adverse effects. To address these challenges, we devised a gene regulation system called Expression Attenuation via Construct Tuning (EXACT), which uses a self-contained, microRNA-based feed-forward loop that not only ensures more consistent transgene expression but also protects against excessive expression. Through cell-based screening assays, we demonstrated the ability of the EXACT circuit to modulate the expression of full-length human MeCP2. Compared with a conventional construct, an EXACT-MECP2 construct exhibited a narrower range of cellular protein abundance. Furthermore, the degree of regulation by the EXACT circuit increased with higher transgene doses in vitro and in wild-type mice and mice modeling RTT. On the basis of cellular and in vivo testing, we identified an optimal configuration for the adeno-associated virus serotype 9 (AAV9) construct for self-regulated MECP2 gene therapy, designated NGN-401. Delivery of NGN-401 to neonatal male Mecp2-/y hemizygous mice via intracerebroventricular injection resulted in prolonged survival and amelioration of RTT-like phenotypes compared with vehicle-treated animals. NGN-401 was also well tolerated by female Mecp2+/- mice and healthy juvenile nonhuman primates, in contrast with a conventional construct, which caused toxicity. The results from these studies underpin a first-in-human pediatric trial of NGN-401 in RTT (ClinicalTrials.gov, NCT05898620).

Animals↗