Biomedical subjects

L Blanchard

Publications and source records attributed to L Blanchard.

At least 19 recordsLinked to original sources

PubMed1998

1H-NMR study of the structural influence of Y64 substitution in Desulfovibrio vulgaris Hildenborough cytochrome c553.

Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical-shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64-->F substitution induces particular effects on the NH exchange at the N-terminal, C-terminal and central alpha-helices and increases the stability of the oxidized molecule.

Amino Acid Sequence↗

PubMed1998

Response latencies for false memories: gist-based processes in normal aging.

Three experiments have demonstrated that age-related increases in both probability and speed of false recognitions for word lists depended on the use of a gist-based memory strategy. When test conditions promoted a gist strategy, both younger and older participants were as likely to falsely recognize a thematically associated lure as to correctly recognize a studied item, and both groups were equally fast in making these decisions. However, when test conditions deemphasized a gist-based strategy, older adults were more likely than younger adults, and faster, to falsely recognize both strong and weakly associated lures. These findings suggest an age-related increase in reliance on gist-based processing that may underlie age differences in false memory.

Adult↗

PubMed1998

Level of ICAM-1 surface expression on virus producer cells influences both the amount of virion-bound host ICAM-1 and human immunodeficiency virus type 1 infectivity.

Using virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.

Cell Line↗

PubMed1997

The effect of ring currents on carbon chemical shifts in cytochromes.

Calculations suggest that some carbon chemical shifts in proteins should have large ring current shifts (> 1 ppm). We present 13C, 15N and 1H assignments for cytochrome C2 from Rhodospirillum rubrum, compare these with shifts for other cytochromes c, and show that the calculated ring current shifts are similar to experimentally observed shifts, but that there remain substantial conformation-dependent shifts of side-chain carbons. Ring current shifts as large as 6 ppm are observed. We show that the ring current effects do not seriously affect the Chemical Shift Index method for delineating secondary structure, but may have an impact on more precise methods for generating structural constraints.

Amino Acid Sequence↗

PubMed1997

Achromatic visual backward masking of colored stimuli in type I diabetes.

On a visual backward masking task using color stimuli with an achromatic patterned mask, we compared the masking performances of 3 Type I diabetics with those of 9 participants in a control group. Analysis indicated that the diabetics show a marked decrement in performance with blue stimuli and a lesser decrement with red stimuli. Suggestions for further theoretical and parametric studies are discussed.

Adult↗

PubMed1996

New conformational properties induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough ferricytochrome c553 using isotopic exchanges monitored by mass spectrometry.

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

Cytochrome c Group↗

PubMed1996

Investigation of oxidation state-dependent conformational changes in Desulfovibrio vulgaris Hildenborough cytochrome c553 by two-dimensional H-NMR spectra.

Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) was used to assign the proton resonances of ferricytochrome C553 from Desulfovibrio vulgaris Hildenborough. The spin systems of 76 out of 79 amino acids were identified by J-correlation spectroscopy (COSY and HOHAHA) in H20 and D20 and correlated by nuclear Overhauser effect spectroscopy (NOESY). The proton chemical shifts are compared in both oxidized and reduced states of the protein at 23 degrees C and pH 5.9. Chemical shift variations between reduced and oxidized states are due to the paramagnetic contribution. Medium and long-range nOe demonstrate the lack of major changes between the two redox states. NMR data provide evidence that in this low oxidoreduction potential cytochrome, the oxidized state is more rigid than the reduced state.

Amino Acid Sequence↗

PubMed1996

Study of the new stability properties induced by amino acid replacement of tyrosine 64 in cytochrome C553 from Desulfovibrio vulgaris Hildenborough using electrospray ionization mass spectrometry.

Hydrogen/deuterium exchange as well as charge state distribution monitored by electrospray ionization mass spectrometry were demonstrated to be a powerful and effective new tool for probing conformational properties of proteins in solution. In this paper, the influence of single amino acid replacements on the global conformation of cytochrome C553 from Desulfovibrio vulgaris Hildenborough using isotopic exchange monitored by electrospray ionization mass spectrometry is reported. Based on their respective charge state distributions and isotopic exchanges, we have differentiated relative stability of mutants and a ladder classification with the order being wild-type > Y64F = Y64L > Y64V > Y64A, under specific conditions of pH, is proposed.

Amino Acid Sequence↗

PubMed1996

Phototoxicity of some bromine-substituted rhodamine dyes: synthesis, photophysical properties and application as photosensitizers.

The synthesis of some bromine-substituted rhodamine derivatives viz., 4,5-dibromorhodamine methyl ester (dye 2) and 4,5-dibromorhodamine n-butyl ester (dye 3) are reported. These dyes were synthesized to promote a more efficient cancer cell photosensitizer for potential use in in vitro bone marrow purging in preparation for autologous bone marrow transplantation. Spectroscopic and photophysical characterization of these dyes together with rhodamine 123 (dye 1) are reported in water, methanol, ethanol and also in a microheterogeneous system, sodium dodecyl sulfate. The possible mechanism of photosensitization is characterized in terms of singlet oxygen efficiency of these dyes. Singlet oxygen quantum yields for bromine-substituted dyes are in the range of 0.3-0.5 depending on the solvent. For dye 1 no singlet oxygen production is found. The photodynamic actions of these dyes in different cell lines are tested. It was found that dye 2 and dye 3 are efficient photosensitizers and mediate eradication of K562, EM2, myeloid cell lines (CML) and the SMF-AI rhabdomyosarcoma line.

Animals↗

PubMed1995

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough.

Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83,500 Da) is proposed to contain a molybdenum cofactor, a 27,000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14,000 Da) holds a c-type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.

Amino Acid Sequence↗

PubMed1995

Control of the redox potential in c-type cytochromes: importance of the entropic contribution.

The enthalpic and entropic components of the redox free energy variation of cytochrome c553 from Desulfovibrio vulgaris Hildenborough and its mutant Y64V, flavocytochrome b2 from Saccharomyces cerevisiae, and the different hemes of cytochromes c3 from Desulfovibrio vulgaris Miyazaki and Desulfovibrio desulfuricans Norway have been determined in 0.1 M Tris-HCl pH 7.0 (7.6 for cytochromes c3) at 25 degrees C by using nonisothermal potentiometric titrations. The set of available experimental data demonstrates that the entropic component plays an important role in the control of the redox potential in c-type and b-type cytochromes. The variation of the entropic component within the class of cytochromes characterized by a positive value of E degrees ' is proposed to be mainly determined by the variation of the exposure of the heme propionates to the solvent. In the case of tetraheme cytochromes c3, the thermodynamic characteristics vary largely among the hemes belonging to the same molecule, which reflects the environmental peculiarities of each heme and also the heme-heme redox interactions. This study substantiates the existence of compensatory effects between large and opposite contributions to E degree ' predicted by all the current theoretical models which are based on electrostatic free energy calculations.

Animals↗

PubMed1995

Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species.

Steady state and time-resolved fluorescence spectroscopy were employed to study the fluorescence from non-metastatic, metastatic and non-tumorigenic cell lines from different species. Excitations at 310 nm and 350 nm were used to monitor tryptophan and reduced nicotinamide adenine dinucleotide (NADH) fluorescence respectively. Subtle and consistent differences were observed between different categories of cell lines. It was found that the tryptophan to NADH fluorescence intensity ratio is higher in metastatic cell lines than in non-metastatic and normal cell lines. The fluorescence decay of the tryptophan residue in different cell lines was best described by triple exponential kinetics, whereas the NADH fluorescence decay was best described by mainly double and, in some cases, triple exponential kinetics. The average fluorescence lifetimes for tryptophan were in the range 2.5-3.7 ns. The average lifetime of NADH was lower (by a factor of approximately three) in metastatic cells than in non-metastatic cells and this finding is consistent for cell lines from different origins (rat or human). Correcting the fluorescence intensity for the average fluorescence lifetime of each species and for the volume of each cell line, it was shown that the concentrations of tryptophan and NADH are consistently higher in malignant metastatic cancer cells than in non-metastatic cells.

Animals↗

PubMed1994

Effects of the Tyr64 substitution on the stability of cytochrome c553, a low oxidoreduction-potential cytochrome from Desulfovibrio vulgaris Hildenborough.

Cytochrome c553 from sulfate-reducing bacteria is a low-oxidoreduction-potential cytochrome. The primary and tertiary structures show notable differences when compared to mitochondrial cytochromes. Tyr64 replacement in cytochrome c553 provides evidence that this residue is not directly involved in the potential modulation but is mostly implicated in the hydrogen-bond network around the heme. While the different variants obtained did not induce drastic structural modifications, they did affect the stability of the protein. This decrease of stability in acidic and alkaline environments was observed by variations in the optical spectra and by mass spectrometry. In addition, the mobility of aromatic side-chain was found to be increased in the mutant proteins as monitored by two-dimensional NMR spectroscopy.

Base Sequence↗

PubMed1994

N-Phosphonomethyl dipeptides and their phosphonate prodrugs, a new generation of neutral endopeptidase (NEP, EC 3.4.24.11) inhibitors.

Inhibitors of the zinc protease neutral endopeptidase (NEP, EC 3.4.24.11) offer significant therapeutic interest as antihypertensives due to their ability to potentiate the biological action of the circulating natriuretic hormone ANF (atrial natriuretic factor). N-Phosphonomethyl dipeptides bearing a central (4-phenyl)phenylalanine residue have been designed to exert potent and selective NEP inhibition. In particular, (S)-3-[N-[2- [(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic acid (10a) (CGS 24592) displayed high inhibitory potency in vitro (IC50 = 1.9 +/- 0.1 nM) and a long plasma half-life in rats but lacked oral bioavailability. This drawback was overcome by using esterase-sensitive (acyloxy)alkyl phosphonates. More remarkable, several diaryl phosphonate derivatives of 10a also performed as effective prodrugs. Specifically, the structurally simple diphenyl phosphonate 18 (CGS 25462) induced potent inhibition of NEP ex vivo for at least 8 h after oral administration to rats (30 mg/kg). Its antihypertensive effect was demonstrated in DOCA-salt rats. At 30 mg/kg orally, 18 caused a significant reduction in mean arterial pressure measuring -35 +/- 7 mmHg at 5-h postdosing. The alpha-aminomethyl phosphonate 18 represents a new generation of selective NEP inhibitors that combine high potency, long duration of action, and oral bioavailability. Therefore, it holds promise as a novel therapeutic agent for the treatment of human hypertension and congestive heart failure.

Animals↗

PubMed1994

The protein moiety modulates the redox potential in cytochromes c.

Cytochrome c is one of the most thoroughly documented oxidoreduction proteins. Its electron transfer activity, which involves an association between the heme group and the polypeptidic chain, is correlated with the redox potential value of the heme group. The redox potential covers a wide range up to 0.8 V, an extreme case being observed in the low-potential cytochromes c from sulfate reducing bacteria. On of the main roles of the polypeptidic moiety consists of modulating the redox potential value of the heme group. In this paper, some structural factors that seem likely to be involved in maintaining the redox potential value are described.

Cytochrome c Group↗

PubMed1993

Overexpression of Desulfovibrio vulgaris Hildenborough cytochrome c553 in Desulfovibrio desulfuricans G200. Evidence of conformational heterogeneity in the oxidized protein by NMR.

Plasmid pRC41, containing the cyf gene encoding cytochrome c533 from Desulfovibrio vulgaris Hildenborough, was transferred by conjugation from Escherichia coli to Desulfovibrio desulfuricans G200. The structural properties of the purified protein were studied by one-dimensional and two-dimensional NMR. A heterogeneity in the folding of the cytochrome isolated from D. vulgaris Hildenborough and from D. desulfuricans G200 was observed for the oxidized from. Temperature, pH and salt-dependence studies indicated that the heterogeneity does not result from an intermediate in the protein unfolding process, but derives from two conformations which are not in dynamic equilibrium.

Amino Acid Sequence↗

PubMed1993

Intramolecular electron transfer in ferredoxin II from Desulfovibrio desulfuricans Norway.

In order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms.

Amino Acid Sequence↗

PubMed1993

Characterization of a lysine-to-glutamic acid mutation in a conservative sequence of farnesyl diphosphate synthase from Saccharomyces cerevisiae.

The mutant gene erg20-2 was isolated from a yeast strain defective in farnesyl diphosphate synthase (FPPS). This strain had the unusual property of excreting prenyl alcohols such as geraniol. The nucleotide (nt) sequence, compared with that of the wild-type gene, showed a single nt change, resulting in a Lys197-->Glu substitution in FPPS which is directly involved in terpenic alcohol formation. In addition, disruption of ERG20 revealed that in yeast no other prenyl transferase is able to synthesize the FPP molecules required for essential non-sterol metabolites.

Acyclic Monoterpenes↗