Biomedical subjects

J R Guest

Publications and source records attributed to J R Guest.

At least 19 recordsLinked to original sources

PubMed2005

Landau quantization and time dependence in the ionization of cold, strongly magnetized Rydberg atoms.

The electric-field-ionization and autoionization behavior of cold Rydberg atoms of 85Rb in magnetic fields up to 6 T is investigated. Multiple ionization potentials and field-ionization bands reflecting the Landau energy quantization of the quasifree Rydberg electron are observed. The time-resolved and state-selective field-ionization study provides evidence of mixing and spin flips of the Rydberg electron. Spin-orbit coupling combined with mixing gives rise to a Feshbach-type autoionization of metastable positive-energy atoms.

Journal Article↗

PubMed2005

Magnetic trapping of long-lived cold Rydberg atoms.

We report on the trapping of long-lived strongly magnetized Rydberg atoms. 85Rb atoms are laser cooled and collected in a superconducting magnetic trap with a strong bias field (2.9 T) and laser excited to Rydberg states. Collisions scatter a small fraction of the Rydberg atoms into long-lived high-angular momentum "guiding-center" Rydberg states, which are magnetically trapped. The Rydberg atomic cloud is examined using a time-delayed, position-sensitive probe. We observe magnetic trapping of these Rydberg atoms for times up to 200 ms. Oscillations of the Rydberg-atom cloud in the trap reveal an average magnetic moment of the trapped Rydberg atoms of approximately -8microB. These results provide guidance for other Rydberg-atom trapping schemes and illuminate a possible route for trapping antihydrogen.

Journal Article↗

PubMed2005

Laser cooling and magnetic trapping at several tesla.

Laser cooling and magnetic trapping of (85)Rb atoms have been performed in extremely strong and tunable magnetic fields, extending these techniques to a new regime and setting the stage for a variety of cold atom and plasma experiments. Using a superconducting Ioffe-Pritchard trap and an optical molasses, 2.4 x 10(7) atoms were laser cooled to the Doppler limit and magnetically trapped at bias fields up to 2.9 T. At magnetic fields up to 6 T, 3 x 10(6) cold atoms were laser cooled in a pulsed loading scheme. These bias fields are well beyond an order of magnitude larger than those in previous experiments. Loading rates, molasses lifetimes, magnetic-trapping times, and temperatures were measured using photoionization and electron detection.

Journal Article↗

PubMed2002

Tunneling resonances and coherence in an optical lattice.

The center-of-mass quantization of atoms trapped in a gray optical lattice is observed to manifest itself in the steady-state properties of the atoms. Modulations in the lifetime and macroscopic magnetization as a function of an applied B field are attributed to quantum mechanical tunneling resonances and are shown to exist only under conditions which afford spatial coherence of the trapped atoms over several lattice wells and coherence times that exceed the tunneling period.

Journal Article↗

PubMed2002

Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals.

AIMS: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia). METHODS AND RESULTS: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases. CONCLUSIONS: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.

Adaptation, Physiological↗

PubMed2001

Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.

Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Amino Acid Sequence↗

PubMed2001

Near-field coherent spectroscopy and microscopy of a quantum dot system.

We combined coherent nonlinear optical spectroscopy with nano-electron volt energy resolution and low-temperature near-field microscopy with subwavelength resolution (<lambda/2) to provide direct and local access to the excitonic dipole in a semiconductor nanostructure quantum system. Our technique allows the ability to address, excite, and probe single eigenstates of solid-state quantum systems with spectral and spatial selectivity while simultaneously providing a measurement of all the various time scales of the excitation including state relaxation and decoherence rates. In analogy to scanning tunneling microscopy measurements, we can now map the optical local density of states of a disordered nanostructure. These measurements lay the groundwork for studying and exploiting spatial and temporal coherence in the nanoscopic regime of solid-state systems.

Journal Article↗

PubMed2001

High-angular-momentum states in cold Rydberg gases.

Cold, dense Rydberg gases produced in a cold-atom trap are investigated using spectroscopic methods and time-resolved electron counting. Optical excitation on the discrete Rydberg resonances reveals long-lasting electron emission from the Rydberg gas ( >20 ms). Our observations are explained by lm-mixing collisions between Rydberg atoms and slow electrons that lead to the population of long-lived high-angular-momentum Rydberg states. These atoms thermally ionize slowly and with large probabilities.

Journal Article↗

PubMed2001

The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives.

The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe(3+) and Zn(2+) derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin. The binding of both metals is accompanied by small changes in the amino acid ligand positions. Mean Fe(A)(3+)-Fe(B)(3+) and Zn(A)(2+)-Zn(B)(2+) distances are 3.24 A and 3.43 A, respectively. In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation. The Fe(3+) derivative alone shows a third metal site (Fe( C)( 3+)) joined to Fe(B)(3+) by a long anti-anti bidentate bridge through Glu130 (mean Fe(B)(3+)-Fe(C)(3+) distance 5.79 A). The third metal site is unique to the non-haem bacterial ferritins. The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water. Models representing the possible binding mode of dioxygen to the dinuclear Fe(3+) pair suggest that a gauche micro-1,2 mode may be preferred stereochemically. Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles. The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s. deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %). Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions.

Amino Acid Sequence↗

PubMed2001

Functional versatility in the CRP-FNR superfamily of transcription factors: FNR and FLP.

The cAMP receptor protein (CRP; sometimes known as CAP, the catabolite gene activator protein) and the fumarate and nitrate reduction regulator (FNR) of Escherichia coli are founder members of an expanding superfamily of structurally related transcription factors. The archetypal CRP structural fold provides a very versatile mechanism for transducing environmental and metabolic signals to the transcription machinery. It allows different functional specificities at the sensory, DNA-recognition and RNA-polymerase-interaction levels to be 'mixed and matched' in order to create a diverse range of transcription factors tailored to respond to particular physiological conditions. This versatility is clearly illustrated by comparing the properties of the CRP, FNR and FLP (FNR-like protein) regulators. At the sensory level, the basic structural fold has been adapted in FNR and FLP by the acquisition in the N-terminal region of different combinations of cysteine or other residues; which bestow oxygen/redox sensing mechanisms that are poised according to the oxidative stress thresholds affecting the metabolism of specific bacteria. At the DNA-recognition level, discrimination between distinct but related DNA targets is mediated by amino acid sequence modifications in the conserved core contact between the DNA-recognition helix and target DNA. And, at the level of RNA-polymerase-interaction, different combinations of three discrete regions contacting the polymerase (the activating regions) are used for polymerase recruitment and promoting transcription.

Amino Acid Sequence↗

PubMed2001

A novel promoter architecture for microaerobic activation by the anaerobic transcription factor FNR.

The yfiD gene of Escherichia coli has an unusual promoter architecture in which an FNR dimer located at -93.5 inhibits transcription activation mediated by another FNR dimer bound at the typical class II position (-40.5). In vitro transcription from the yfiD promoter indicated that FNR alone can downregulate yfiD expression. Analysis of yfiD::lac reporters showed that five turns of the DNA helix between FNR sites was optimal for downregulation. FNR heterodimers, in which one subunit carried a defective repression surface, revealed that the upstream subunit of the -40.5 dimer and the downstream subunit of the -93.5 dimer were most important for downregulating yfiD expression. Deletion of the C-terminal domain of the alpha-subunit of RNA polymerase (RNAP) did not affect FNR-mediated repression, suggesting that repression is mediated through FNR-FNR and not FNR-RNAP interactions. Maximum yfiD::lac expression was observed in cultures exposed to 10 microM oxygen. More or less oxygen reduced expression dramatically. This pattern of response was dependent on the combination of a high-affinity site at the activating class II position and a lower affinity site at the upstream position.

Aerobiosis↗

PubMed2001

Anaerobic acquisition of [4FE 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions.

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia. The anaerobic incorporation of oxygen-sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding. Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen-sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway. The C122 variant failed to assemble iron-sulphur clusters and to bind DNA. Second-site substitutions that partially restore activity to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was concluded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.

Amino Acid Substitution↗

PubMed2000

Ponderomotive optical lattice for Rydberg atoms.

We propose to use the ponderomotive energy of Rydberg electrons in standing-wave light fields to form an optical lattice for Rydberg atoms. Application of the Born-Oppenheimer approximation shows that, with readily achievable experimental parameters, atoms in any Rydberg state can be trapped. Realization of this scheme would extend the benefits of atom trapping to highly excited atoms.

Journal Article↗

PubMed2000

Zinc uptake, oxidative stress and the FNR-like proteins of Lactococcus lactis.

Lactococcus lactis ssp. cremoris MG1363 contains two FNR homologues, FlpA and FlpB, encoded by the distal genes of two paralogous operons (orfX(A/B)-orfY(A/B)-flpA/B). An flpA flpB double mutant strain is hypersensitive to hydrogen peroxide and has a depleted intracellular Zn(II) pool. The phenotypes of the flp mutant strains suggest that FlpA and FlpB control the expression of high and low affinity ATP-dependent Zn(II) uptake systems, respectively. Plate tests revealed that expression from a orfX(B)::lac reporter was activated by Cd(II), consistent with other Zn(II)-regulated systems. The link between a failure to acquire Zn(II) and hypersensitivity to oxidative stress suggests that Zn(II) may be required to protect vulnerable protein thiols from oxidation.

Adenosine Triphosphate↗

PubMed2000

Role of activating region 1 of Escherichia coli FNR protein in transcription activation at class II promoters.

FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the -35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase alpha-subunit. Using a model class II FNR-dependent promoter, FF(-41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.

Bacterial Proteins↗

PubMed2000

Characterization of the Lactococcus lactis transcription factor FlpA and demonstration of an in vitro switch.

The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST-FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen-responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA') with an N-terminal region designed to be more FLP-like failed to incorporate an iron-sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen-labile [4Fe 4S] containing FNR-like protein into a dithiol-disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA-based aerobic-anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR-dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.

Amino Acid Sequence↗

PubMed1999

Biochemical and spectroscopic characterization of Escherichia coli aconitases (AcnA and AcnB).

Escherichia coli contains two major aconitases (Acns), AcnA and AcnB. They are distantly related monomeric Fe-S proteins that contain different arrangements of four structural domains. On the basis of the differential expression of the acnA and acnB genes, AcnA has been designated as an aerobic-stationary-phase enzyme that is specifically induced by iron and oxidative stress, whereas AcnB functions as the major citric-acid-cycle enzyme during exponential growth. The biochemical and kinetic properties of the purified enzymes have now shown that AcnA is more stable than AcnB, has a higher affinity for citrate, and operates optimally over a wider pH range, consistent with its role as a maintenance or survival enzyme during nutritional or oxidative stress. In contrast, the better performance at high substrate concentrations and greater instability of AcnB indicate that AcnB is specifically adapted to function as the main catabolic enzyme and, by inactivation, to rapidly modulate energy metabolism in response to oxidative or pH stress, either directly or indirectly by regulating post-transcriptional gene expression. EPR and magnetic-CD spectroscopy showed that the iron-sulphur clusters of the bacterial Acns (and their binding sites) strongly resemble those of the mammalian enzymes. The EPR and MCD spectra of the oxidized inactive form of AcnB confirmed the presence of a [3Fe-4S](1+) (S=1/2) cluster. Comparisons showed that the EPR spectrum of AcnB more closely resembled that of mammalian mitochondrial Acn (m-Acn), whereas the spectrum of AcnA more closely resembled that of the cytoplasmic enzyme (c-Acn). The MCD spectra revealed spectroscopic signatures similar to that of m-Acn. Reconstitution of the active [4Fe-4S](2+) forms followed by one-electron reduction gave rise to EPR spectra that are almost identical with those reported for the mammalian enzymes.

Aconitate Hydratase↗

PubMed1999

Two operons that encode FNR-like proteins in Lactococcus lactis.

Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products. Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR-like proteins (FlpA and FlpB) with the potential for mediating a dithiol-disulphide-dependent regulatory switch, were identified. Transcript analysis indicated that they are distal genes of two paralogous operons, orfX-orfY-flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively. The corresponding promoters were each associated with a potential FNR site (TTGAT----ATCAA) at positions +4.5 (flpA operon) and -42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated. The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases. No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two-dimensional gel electrophoresis): formate production was not affected. It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.

Amino Acid Sequence↗