Biomedical subjects

M Panda

Publications and source records attributed to M Panda.

18 recordsLinked to original sources

PubMed2005

Recruitment of governing elements for electron transfer in the nitric oxide synthase family.

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O(2) was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.

Animals↗

PubMed2002

Structural effects on the formation of proton and alkali metal ion adducts of apolar, neutral peptides: electrospray ionization mass spectrometry and Ab initio theoretical studies.

Apolar, neutral peptides have been shown to ionize extremely well under the conditions used for electrospray ionization mass spectrometry (ESIMS). Peptides for which the conformations have been independently determined in solution and in crystals have been examined by ESIMS. Studies of peptide helices ranging from 7 to 18 residues reveal that shorter helices yield exclusively singly charged ions, while in larger helices multiply charged species are detectable. Multiple sites for protonation/metallation are introduced in the helix by proline insertion or by changing the chirality in the residue. The preferred site of cation binding to helices may be the C-terminus end, where three free CO groups are available for chelation. Ab initio and DFT calculations at several levels have been carried out for the binding of H+, Li+, Na+, and K+ to CHO-(Gly)3)-OMe. The results reveal that metallation in helices is favoured by chelation to carbonyl groups at the C-terminus, while protonation involved two carbonyl groups and thus favour a 10-membered cyclic hydrogen-bonded structure. In -strands, metallation/protonation occurs at isolated carbonyl groups. Collision induced fragmentation of hydrophobic peptides under ESI conditions reveals that helix fragmentation occurs predominantly from the C-terminus, while in -hairpins cleavage occurs simultaneously at multiple sites.

Amino Acids, Neutral↗

PubMed2001

The 4,4'-dipyridyl disulfide-induced formation of GroEL monomers is cooperative and leads to increased hydrophobic exposure.

The molecular chaperone, GroEL, is completely disassembled into monomers by the addition of 4,4'-dipyridyl disulfide. The dissociation leads to monomers in a kinetically controlled process. The additions of functional ligands of GroEL such as Mg(2+) or adenine nucleotides produced differences in the observed rates, but at the end of the kinetics, the dissociation was complete. In addition to the information obtained from native gels, the fluorescent probe bis-ANS was utilized to follow the monomer formation. The results demonstrate that the formation of monomers was associated with the exposure of hydrophobic surfaces. This assessment was possible without the use of added chaotropes, such as urea, to dissociate GroEL. Dissociation kinetics were also followed by light scattering. The kinetics of dissociation of the 14mer are cooperative with respect to the concentration of 4,4'-DPDS. Thermodynamic parameters for the kinetic process gave a free energy of activation (DeltaG) of 19.3 +/- 1.2 kcal mol(-1), which was decomposed to an enthalpy of activation (DeltaH) of 19.30 +/- 1.2 kcal mol(-1) and an entropy of activation (DeltaS) of -8.2 +/- 3.9 cal mol(-1) K(-1). We conclude that the dissociation of GroEL observed in this investigation is an enthalpy-controlled process.

Adenine Nucleotides↗

PubMed2001

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.

Anilino Naphthalenesulfonates↗

PubMed2000

High hydrostatic pressure can probe the effects of functionally related ligands on the quaternary structures of the chaperonins GroEL and GroES.

We investigated the effects of high hydrostatic pressure in the range of 1--3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg(2+), ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg(2+) and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1--2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system.

Adenosine Triphosphate↗

PubMed2000

Productive and nonproductive intermediates in the folding of denatured rhodanese.

The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 M urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species.

Animals↗

PubMed2000

Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese.

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM beta-ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (N<-->I<-->U) followed by fluorescence at 363 nm, (N<-->I) by fluorescence at 318 nm, and CD (I<-->U) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5-6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.

Binding Sites↗

PubMed2000

Cytochrome c folds through a smooth funnel.

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.

Cytochrome c Group↗

PubMed1999

A bile acid-induced apoptosis assay for colon cancer risk and associated quality control studies.

Bile acids are important in the etiology of colorectal cancer. Bile acids induce apoptosis in colonic goblet cells at concentrations comparable to those found in fecal water after high-fat meals. Preliminary evidence indicated that cells of the normal-appearing (nontumorous) portion of the colon epithelium of colon cancer patients are more resistant to bile salt-induced apoptosis than are cells from normal individuals. In the present study, 68 patients were examined, and biopsies were taken at 20 cm from the anal verge, cecum, and descending colon. The patients included 17 individuals with a history of colorectal cancer, 37 individuals with adenomas, and 14 individuals who were neoplasia free. The mean bile salt-induced apoptotic index among normal individuals was 57.6 +/- 3.47 (SE), which differed significantly (P < 0.05) from the mean value of 36.41 +/- 3.12 in individuals with a history of colon cancer. The correlation between independent observers was 0.89 (P < 0.001), indicating good interobserver reliability. Components of variance comparing interindividual versus intraindividual sources of variation suggested that site-to-site variability, both between regions of the colon and for adjacent biopsies, was larger than the interpatient variability for individuals with a history of neoplasia. Therefore, there was "patchiness" of the susceptibility of regions of the colon to bile acid-induced apoptosis in individuals with a history of neoplasia (a patchy field effect). There was no obvious correlation of low-apoptotic index regions with regions in which previous neoplasias had been found and removed. On the other hand, for normal, i.e., neoplasia-free, individuals, there was relatively less intraindividual variation compared to interindividual variation. Our assay shows an association between resistance to bile acid-induced apoptosis, measured at 20 cm from the anal verge, and colon cancer risk. Thus, this assay may prove useful as a biomarker of colon cancer risk.

Adenoma↗

PubMed1998

Treatment of chronic osteomyelitis using the Papineau technique.

From 1984 to 1994, 41 cases of chronic osteomyelitis were treated in Kinshasa by the Papineau technique; 75.5% were men and 24.5% women. The mean age was 28 years (range: 7-77), and the average duration of the infection was 3 years (range: 1 month to 28 years). The aetiology was "trauma" in 53.5%, haematogenous in 44% and drepanocytosis in 2.5%. Infections of the femur and tibia each constituted 41.5% of the cases. Immobilisation was by external fixation in 44%, and by a cast in 39%. Wound healing was complete after an average of 3 months with spontaneous healing, and in 4.5 months after skin grafting. Control of the infection and bone healing were obtained in 89% after a period of 3-7 months. We discuss the Papineau technique and the modifications which were required in Kinshasa.

Adolescent↗

PubMed1995

Kinetics and mechanism for the binding of HCN to cytochrome c oxidase.

The kinetics of cyanide binding to cytochrome c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k'obs) follows saturation type kinetics to form an HCN complex that subsequently undergoes a slow reaction (k'obs). The fast k'obs reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pKa = 7.45), while the other is that of HCN (pKHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k'obs reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k'obs reaction, the oxidase-HCN complex, still undergoes the slow k" process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k"obs) is independent of all the variations done in this study, and it probably corresponds to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monomeric or dimeric oxidase only slightly affects the magnitude of k'obs and k"obs values, and both processes are clearly present in both types of oxidase.

Animals↗

PubMed1992

A socioeconomic analysis of digital replantations resulting from home use of power tools.

Forty-seven digits in 21 patients who sustained partial or complete amputations from the home use of power tools were replanted or revascularized during a 1-year period. This represented 69% of the microvascular surgery performed for hand injuries during that year. The cost of repairing an average of 2.4 digits was $7000 (surgeon's fee) plus $697 per hour (operating room fee), as per fiscal year 1987. Postoperative hospitalization averaged 15 days at a cost of $15,679. Hand rehabilitation averaged 8 months at a cost of $3348. Fifty-four percent of the patients had no insurance. Fourteen of 21 patients (67%) required at least one additional procedure. Two patients had to make a career change after the injury. The majority of patients with digital replantations were dissatisfied with the emotional costs and the number of subsequent operations. Lack of patient and family awareness of the length of the rehabilitative period was particularly evident.

Accidents, Home↗

PubMed1988

St. Clair Strange procedure: indications, technique, and long-term evaluation.

The pedicled ulnar nerve transfer (St. Clair Strange procedure) preserves the intrinsic blood supply to the transferred nerve and allows bridging of large defects in avascular, scarred wounds. We report our indications for this operation, describe the two operative stages, and report the results in seven patients. After operation progressive median nerve axonal growth in a proximal direction up the ulnar nerve was shown by a median nerve Tinel's sign. Five patients followed for a mean of 10 years achieved protective sensibility in the median nerve distribution. In two of the three patients in whom distal nerve repair was done to the median and ulnar nerve, protective sensibility also returned to the ulnar digits. Localization, stereognoses, and vibratory response was achieved in the majority of digits. All patients had active use of the hand, but none had meaningful two-point discrimination. No changes were found after a superficial radial nerve block excluded any radial nerve contribution. No intrinsic motor functional return was noted. Two patients are currently being followed.

Adolescent↗

PubMed1977