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F Schaeffer

Publications and source records attributed to F Schaeffer.

17 recordsLinked to original sources
PubMed1999

Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry.

When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs.

Animals↗
PubMed1999

Development of a computer based training program for liver transplantation.

The existence and future of every scientific area is directly and indirectly dependent on advanced education in that area and the quality of the work performed. Our aim in developing this program was to improve advanced education in liver transplant surgery and to ensure the quality of the work performed by every surgeon operating in this field. We developed a module that contains extensive information on the pathogenesis, symptoms, diagnosis, X-rays and treatment plans as well as operation-videos and case studies on the most common aspects in liver transplantation. This "teachware" is now on CD-ROM and can be installed on every personal computer without special computer knowledge. The expansion and updating of the material is technically facilitated with the help of an authoring tool.

CD-ROM↗
PubMed1998

Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa.

Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2 and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincubation of hsPLA2 with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a Kd of 230 nM. By using surface plasmon resonance we determined the rate constants, kon and koff, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51-74 and 51-62 of hsPLA2 inhibited prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51-62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the prothrombinase generation by preventing FXa.FVa complex formation.

Amino Acid Sequence↗
PubMed1997

DNA curvature controls termination of plus strand DNA synthesis at the centre of HIV-1 genome.

In vivo and in vitro, reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) terminates plus strand synthesis at the centre of the viral genome. The central termination sequence (CTS) contains curved DNA fragments located upstream of each terminator site. Two different models, relying either on the A-tract or general sequence roll assumptions, were used to predict the extent and the direction of this curvature as well as to design mutants, which abolished it. Straightening of each curved element abolished termination at the site located immediately downstream from the curvature. When synthesis was performed on the other strand and in the opposite direction, the two curved elements C1 and C2 associated with the two termination sites Ter1 and Ter2, led again to termination of DNA synthesis. Therefore, termination occurred as a nascent bent duplex was synthesized within the template primer binding cleft of RT, even when putative strand-specific motifs have been removed by the inversion. Computation of DNA paths upstream of other known arrest sites suggested that this feature was of general relevance for termination. At the CTS, termination occurred more precisely at the 3' end of an AnTm motif (n + m = 7). The possible structures, adopted by this motif, are discussed and confronted with the present crystallographic and biochemical data obtained on HIV-1 RT-DNA interactions and on HIV-1 RT processivity.

Base Sequence↗
PubMed1997

Relationships between H-NS, sigma S, SpvR and growth phase in the control of spvR, the regulatory gene of the Salmonella plasmid virulence operon.

The sigma S-regulated gene spvR of Salmonella typhimurium encodes an autoregulatory protein required for transcriptional activation of the virulence operon spvABCD. A mutation in the histone-like protein H-NS, which negatively controls the sigma S level, has been reported to increase spv gene expression in S. typhimurium strain LT2. In agreement with this, we found that transcription of spvR and spvABCD was derepressed in hns strains of Escherichia coli and S. typhimurium. Moreover, levels of spv gene expression in hns rpoS double mutants were higher than expression levels in mutants deficient in rpoS alone, and were close to those measured in wild-type strains. This demonstrates that H-NS contributes to spv gene regulation independently of its function in controlling the sigma S level. Since the same start site was used for spvR gene transcription in wild-type as in hns and hns rpoS mutant strains, it is likely that the spvR promoter. spvRp1, can be recognized efficiently by an RNA polymerase containing sigma 70. The spvR promoter region shows an intrinsic DNA curvature that might be a determinant in H-NS- and/or sigma S-mediated control. A single amino acid substitution, Leu to Pro at position 265, abolished the regulatory function of SpvR in E. coli and Salmonella, implicating the C-terminal domain of SpvR in its structure and/or regulatory function. The spvR265 allele is not transcribed at detectable levels in hns or hns rpoS strains, suggesting that activation of spvRp1 in these strains remains dependent on SpvR. Thus, we propose a model for spvR gene regulation in which SpvR acts as a co-regulator of an RNA polymerase containing either sigma 70 (in the absence of H-NS) or sigma S, to induce transcriptional initiation at spvRp1. Moreover, growth-phase regulation of spv gene expression was maintained in hns and hns rpoS strains, indicating that an additional element, besides sigma S, is involved in the growth-phase regulation in rich medium.

Bacterial Proteins↗
PubMed1996

DNA-stacking interactions determine the sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.

Bis(1,10-phenanthroline)-copper(I) ion (OP2Cu+) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-dependent yet not nucleotide-specific at the cutting site. OP2Cu+ sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (AnTm/AmTn, CnTmCp/GpAmGn), alternating purine-pyrimidine tracts ((TA)n/(TA)n, (TG)n/(CA)n) and at certain G+C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP2Cu+ binding and cleavage specificity based on the free-energy equality is proposed.

Base Composition↗
PubMed1995

Human immunodeficiency virus type 1 reverse transcriptase tG:T mispair formation on RNA and DNA templates with mismatched primers: a kinetic and thermodynamic study.

The relationship between human immunodeficiency virus (HIV) type 1 reverse transcriptase tG:T mispair formation and base pair stability was investigated using DNA and RNA templates with 15 bp matched or mismatched DNA primers. tG:T mispair formation during primer elongation was undetectable on tDNA-DNA duplexes but occurred with a frequency of 10(-4) on matched tRNA-DNA duplexes. The frequency increased to 7.0 x 10(-4) and 1.3 x 10(-3) on tRNA-DNA duplexes with tG:T mismatches located 6 and 9 bp beyond the polymerization site. From Km values at 37 degrees C, the free energy change upon dissociation (delta G degrees 37) of the tG:T mispair increased from matched to mismatched tRNA-DNA duplexes by 0.36-1.21 kcal/mol. delta G degrees 37 for a correct tG:C pair decreased by 0.06-1.00 kcal/mol. In comparison with DNA-DNA duplexes, thermal melting measurements on RNA-DNA duplexes demonstrated smaller enthalpy (delta delta H degrees = -17.7 to -28.1 kcal/mol) and entropy (delta delta S degrees = -59.3 to -83.4 cal/mol/K) components. A strong entropy-enthalpy compensation resulted in small free energy differences (delta delta G degrees 37 = 0.8 to -2.2 kcal/mol). Thus, although DNA-DNA and RNA-DNA duplexes are of comparable stability in solution, the RNA-DNA duplex presents more facile base pair opening and higher conformational flexibility. The release of helical strain at constant helix stability in RNA-DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G-->A hypermutation.

Base Composition↗
PubMed1989

[A persistent case of kennel cough caused by Filaroides osleri].

A dog showing chronic symptoms of cough, which was treated for kennel cough for nearly twelve months, was found to be infected with Filaroides osleri. A single course of treatment with 0.2 mg/kg of ivermectin was followed by subsidence of the symptoms which disappeared within a fortnight.

Animals↗
PubMed1989

Long-range structural effects in supercoiled DNA: statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion.

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817-827] a set of sequences, called C-type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C-type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S-type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations: 1. the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C-type character; 2. C-type induction by an A + T rich Drosophila sequence; 3. low-temperature cruciform extrusion by an (AT)34 sequence; 4. the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat; 5. the effects of systematic deletion of the colL sequence; and 6. the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat. These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.

Base Composition↗
PubMed1989

A rigorous mathematical treatment for the excluded volume effect in Monte Carlo simulations of polymeric chains.

In Monte Carlo simulations of polymeric chains, the chains are most often represented as spheres, or cylinders with flat ends. In this methodological paper, we adopt a representation of the chains as spherocylinders (continuous cylinders ending in semispheres). With such a representation the testing for chain overlap, which is the crucial step for the inclusion of the excluded volume effect in the simulations, can be defined in a rigorous geometrical framework. The treatment we then derive fulfills the following features: it allows a very simple, automatic, and exhaustive classification of all the possible configurations; and it provides a physical representation for steric hindrance effects more natural than the flat-ended cylinders. Notably, this representation avoids the introduction of artificial anisotropies in the treatments. This spherocylindrical representation is also well suited for several types of calculations that can be involved in elaborate Monte Carlo simulations.

Biopolymers↗
PubMed1989

[Therapeutic problems of intra-abdominal liposarcoma].

Intra-abdominal liposarcomas are very rare tumors. We present our personal experience with one case of mesenteric liposarcoma which had perforated into the peritoneal cavity, occurring in a seven-month pregnant female patient. Seven years later, the patient is doing well. The authors have carried out a review of the literature. In addition to liposarcomas of the mesentery and the mesocolon, they observed some cases of liposarcoma localized in the omentum and the stomach; other abdominal localizations occurred sporadically. Radical surgical excision was the choice treatment. Both the propensity for locoregional spread and the histological diversity set the conditions for prognosis; the latter seemed minimally influenced by either radio- or chemotherapy.

Abdominal Neoplasms↗
PubMed1984

The influence of cortisol excess on kidney function in the dog.

The effects of cortisol excess on kidney function were studied in 8 normal conscious dogs. Cortisol was given orally until polyuria developed. Cortisol excess decreased urine osmolality (from 897 +/- 76 to 186 +/- 36 mosm. kg-1) and increased urine production (from 0.7 +/- 0.1 to 9.3 +/- 2.4 ml kg-1. h-1). The glomerular filtration rate increased by 23 +/- 9 per cent. Sodium and potassium concentrations in plasma were decreased. 66 Per cent of the increase in urine production was due to the increase in free water clearance and 34 per cent to the increased urea excretion. Cortisol excess apparently caused polyuria by inhibition of the action of ADH in the collecting duct, resulting in a decreased water and urea reabsorption. The decreased urea reabsorption possibly causes a smaller urea recirculation in the renal medulla and hence a decrease in concentrating capacity.

Animals↗
PubMed1982

Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory.

To understand the denaturation process of short DNA segments we have chosen a 203-base pair (bp) restriction fragment containing the lactose control region. A steady decrease in GC content exists between its i proximal and z proximal ends. We confirm that this fragment melts at low salt in two subtransitions. A GC to AT mutation in the AT-rich region (mutation UV5) increases the number of denatured base pairs in the first subtransition and decreases the cooperativity of the melting process. A GC to AT mutation in the GC-rich region (mutation L8) decreases the number of denatured base pairs in the first subtransition and increases the cooperativity. These mutations induce the same shift in the temperature of half denaturation. The effects of both mutations are additive. A short deletion at the z end of the fragment affects only the first subtransition. When four GC pairs are added to both end, the fragment melts in one transition. Comparison with the results obtained with a larger 789-bp lac fragment reveals strong end effects on base pair stability and suggests that denaturation of the 203-bp fragment proceeds unidirectionally from the z end. Good agreement is shown with the predictions made with the "z ipper model" of Crothers et al. (1965).

Base Sequence↗
PubMed1978

Glucose-6-phosphate dehydrogenase of Anabaena sp. Kinetic and molecular properties.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three pincipal forms, with approximate molecular weights of 120 000 (M1), 240 000 (M2) and 345 000 (M3). The relative catalytic activities are: M1 less than M2 less than M3. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M1 form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP+), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M1 form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M1 form is favored at pH values above 7; a pH of 6.5 is optimal for maintenance of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5'-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.

Adenosine Triphosphate↗