Squamous Cell Carcinoma Arising from Sinonasal Inverted Papilloma.

Sinonasal inverted papillomas occasionally undergo malignant transformation into squamous cell carcinoma, which can be associated with EGFR mutations. Since biopsy can potentially under-sample the tumor, CT and MRI can provide clues as to the presence of malignant transformation. In particular, this entity tends to appear different from benign inverted papilloma on imaging, including prominent bone erosions, necrosis, low diffusivity in the solid tumor components, and absence of the cerebriform pattern on MRI. The radiology findings, pathology features, and management of squamous cell carcinoma arising from inverted papilloma are described.

Synchronization of the acoustic evidence in the assassination of President Kennedy.

We have revisited the acoustic evidence in the Kennedy assassination--recordings of the two Dallas police radio channels upon which our original NRC report (Ramsey NF et al., Report of the Committee on Ballistic Acoustics. National Research Council (US). Washington: National Academy Press, 1982. Posted at http://www.nap.edu/catalog/10264.html) was based--in response to the assertion by DB Thomas (Echo correlation analysis and the acoustic evidence in the Kennedy assassination revisited. Science and Justice 2001; 41: 21-32) that alleged gunshot sounds (on Channel 1), apparently recorded from a motorcycle officer's stuck-open microphone, occur at the exact time of the assassination (as established by emergency communications on Channel 2). We have critically reviewed these two publications, and have performed additional analyses. In particular we have used recorded 60 Hz hum and correlation methods to obtain accurate speed calibrations for recordings made on both channels, cepstral analysis to seek instances of repeated segments during playback of Channel 2 (which could result from groove jumping), and spectrographic and correlation methods to analyze instances of putative crosstalk used to synchronize the two channels. This paper identifies serious errors in the Thomas paper and corrects errors in the NRC report. We reaffirm the earlier conclusion of the NRC report that the alleged "shot" sounds were recorded approximately one minute after the assassination.

The I1-imidazoline receptor in PC12 pheochromocytoma cells activates protein kinases C, extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK).

We sought to further elucidate signal transduction pathways for the I1-imidazoline receptor in PC12 cells by testing involvement of protein kinase C (PKC) isoforms (betaII, epsilon, zeta), and the mitogen-activated protein kinases (MAPK) ERK and JNK. Stimulation of I1-imidazoline receptor with moxonidine increased enzymatic activity of the classical betaII isoform in membranes by about 75% and redistributed the atypical isoform into membranes (40% increase in membrane-bound activity), but the novel isoform of PKC was unaffected. Moxonidine and clonidine also increased by greater than two-fold the proportion of ERK-1 and ERK-2 in the phosphorylated active form. In addition, JNK enzymatic activity was increased by exposure to moxonidine. Activation of ERK and JNK followed similar time courses with peaks at 90 min. The action of moxonidine on ERK activation was blocked by the I1-receptor antagonist efaroxan and by D609, an inhibitor of phosphatidylcholine-selective phospholipase C (PC-PLC), previously implicated as the initial event in I1-receptor signaling. Inhibition or depletion of PKC blocked activation of ERK by moxonidine. Two-day treatment of PC12 cells with the I1/alpha2-agonist clonidine increased cell number by up to 50% in a dose related manner. These data suggest that ERK and JNK, along with PKC, are signaling components of the I1-receptor pathway, and that this receptor may play a role in cell growth.

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.

The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation.

The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 degrees C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120--128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63--70).

An additional serine residue at the C terminus of rhodanese destabilizes the enzyme.

The rhodanese coding sequence was extended at its 3' end by three base pairs to generate mutants coding for a serine or arginine residue at the carboxyl terminus of the protein. Wild-type and mutant coding sequences were expressed in a cell-free Escherichia coli system by coupled transcription/translation. Predominantly full-length protein was formed in all cases. The amount of protein synthesized was quantified by incorporation of radioactive leucine into polypeptides. Enzymatic activity of in vitro synthesized rhodanese was determined at different temperatures. Specific enzymatic activity was calculated and is assumed to reflect the portion of the protein that is in its native three-dimensional conformation. It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incubated above 30 degrees C, in contrast to wild-type protein or variant rhodanese extended by an arginine residue. Similarly, variant rhodanese with an additional serine residue was more susceptible to urea denaturation than the other two rhodanese species. These results are surprising in light of the crystal structure of the protein.

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.

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.

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.

Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding.

The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at approximately 4 m urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5 m urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.

Rhodanese as a thioredoxin oxidase.

A major catalytic difference between the two most common isoforms of bovine liver mitochondrial rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed. Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate. However, only the less negative form in common with the recombinant mammalian rhodanese expressed in E. coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species. These activities are understood in terms of the established persulfide structure (R-S-SH) of the covalently substituted rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R-S-OH) when the enzyme acts as a thioredoxin oxidase. The observations suggest a role for one rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals.

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.

Domain separation precedes global unfolding of rhodanese.

The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.

Nucleotide and Mg2+ induced conformational changes in GroEL can be detected by sulfhydryl labeling.

The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the molecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg2+ and/or adenine nucleotides. Three peptides, each containing one of the cysteines of GroEL (C138, C458 and C519) were identified. GroEL labeled in 50 mM TrisHCl, pH 7.8, incorporated approximately 0.3 labels each on C138 and C458. With 10 mM MgCl2, the labeling increased to approximately 0.8 labels each on C138 and C458. The increase was partially due to a conformational change which occurred upon Mg2+ binding as well as to an increase in ionic strength. When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg2+, C138 incorporated approximately 0.8 labels, while C458 incorporated approximately 0.1 labels. These results suggest that the binding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible. GroEL slowly dissociated into monomers when it was extensively labeled at C458. GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was able to bind but not release active rhodanese. The observed variations in sulfhydryl accessibility are consistent with mechanisms that suggest binding of GroES to GroEL differs from the binding of substrate protein to GroEL, and that the binding of Mg2+ or Mg-adenine nucleotides results in conformational changes in GroEL.

NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions.

The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.

GroES in the asymmetric GroEL14-GroES7 complex exchanges via an associative mechanism.

The interaction of the chaperonin GroEL14 with its cochaperonin GroES7 is dynamic, involving stable, asymmetric 1:1 complexes (GroES7.GroEL7-GroEL7) and transient, metastable symmetric 2:1 complexes [GroES7.GroEL7-GroEL7.GroES7]. The transient formation of a 2:1 complex permits exchange of free GroES7 for GroES7 bound in the stable 1:1 complex. Electrophoresis in the presence of ADP was used to resolve free GroEL14 from the GroES7-GroEL14 complex. Titration of GroEL14 with radiolabeled GroES7 to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex. No stable 2:1 complex was detected. Preincubation of the asymmetric GroES7.GroEL7-GroEL7 complex with excess unlabeled GroES7 in the presence of ADP demonstrated GroES7 exchange. The rates of GroES7 exchange were proportional to the concentration of unlabeled free GroES7. This concentration dependence points to an associative mechanism in which exchange of GroES7 occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL14. Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES7. In agreement with recent structural studies, this indicates that conformational changes in GroEL14 following the dissociation of GroES7 must precede ADP release. These results explain how the GroEL14 cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes.

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression.

Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC. Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.