Biomedical subjects

T Schindler

Publications and source records attributed to T Schindler.

30 records · Page 2Linked to original sources

PubMed1998

Prolyl isomerases do not catalyze isomerization of non-prolyl peptide bonds.

Prolyl isomerases accelerate the cis <--> trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.

Amides↗

PubMed1997

Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product.

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

Acetylation↗

PubMed1997

Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate.

The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.

Amino Acid Isomerases↗

PubMed1997

Diffusion control in an elementary protein folding reaction.

The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N &lrharr; U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.

Bacillus subtilis↗

PubMed1996

Thermodynamic properties of an extremely rapid protein folding reaction.

The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to avoid misfolding prior to the rate-limiting step, and a native-like activated state reduces the risk of non-productive side reactions during the final steps to the native state.

Bacillus subtilis↗

PubMed1996

The role of a trans-proline in the folding mechanism of ribonuclease T1.

Protein folding is often retarded by the cis reversible trans isomerizations of prolyl peptide bonds both in vitro and in vivo. An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolines) by comparing the wild-type protein with the Pro73-->Val variant. The Pro-->Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding. In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged. trans-Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines. The cis-proline residues dominate the kinetics of refolding, because almost all slow-folding molecules contain the respective incorrect (trans) isomers, and because trans-->cis isomerizations are slower than cis-->trans isomerizations. The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this proline is non-essential for folding in the sense that its cis reversible trans isomerization is energetically uncoupled from conformational folding.

Enzyme Stability↗

PubMed1996

Structural rearrangements on HIV-1 Tat (32-72) TAR complex formation.

Expression of the early genes of the human immunodeficiency virus type-I (HIV-1) genome is under the control of a trans-activator (Tat) protein. HIV-1 Tat action requires binding to TAR (trans-activation responsive element), an RNA sequence located at the 5'-end of all lentiviral mRNAs. We used various spectroscopic methods to investigate conformational changes on HIV-1 TAR binding to the HIV-1 (32-72) Tat peptide BP1. It comprises the RNA binding region and binds specifically to TAR. We conclude from our experiments that the regular A-form of the TAR RNA is slightly distorted towards the B-form when bound to BP1. Thus, the major groove is widened and the binding of BP1 facilitated. BP1 presumably adopts an extended conformation when binding to TAR and may fit well into the TAR major groove.

Amino Acid Sequence↗

PubMed1995

Extremely rapid protein folding in the absence of intermediates.

Here we used the cold-shock protein CspB from Bacillus subtilis to study protein folding at an elementary level. The thermodynamic stability of this small five-stranded beta-barrel protein is low, but unfolding and refolding are extremely rapid reactions. In 0.6 M urea the time constant of refolding is about 1.5 ms, and at the transition midpoint (4 M urea) the folded and unfolded forms equilibrate in less than 100 ms. Both the equilibrium unfolding transition and the folding kinetics are perfectly described by a N<-->U two-state model. The validity of this model was confirmed by several kinetic tests. Folding intermediates could neither be detected at equilibrium nor in the folding kinetics. We suggest that the extremely rapid folding of CspB and the absence of folding intermediates are related phenomena.

Bacillus subtilis↗

PubMed1995

Arabinogalactan proteins in maize coleoptiles: developmental relationship to cell death during xylem differentiation but not to extension growth.

The spatial and temporal expression of arabinogalactan proteins (AGPs) in the coleoptile of maize seedlings was investigated with monoclonal antibodies (MAC207, JIM13, JIM14) raised against particular AGP epitopes in carrot. MAC207 binds to a buffer-soluble AGP fraction of 90-210 kDa that also reacts with beta-glucosyl Yariv reagent and the lectin RCA120. Immunogold-labelling showed that the MAC207 epitope is exclusively localized in the plasma membrane. JIM13 binds to a 120 kDa component of the buffer-soluble AGP fraction localized in the plasma membrane of future sclerenchyma cells and secondary-wall thickenings of future tracheids of vascular bundles. JIM14 binds to a 50 kDa component of the salt-extractable fraction from cell walls localized in the innermost wall layer of sclerenchyma cells. These AGP epitopes demonstrate different temporal expression patterns which do not correlate with extension growth. Auxin had no effect on the amount of soluble AGP from coleoptile sections, containing the growth-controlling epidermis but no vascular bundles, as measured by crossed electrophoresis. Moreover, incorporation of radioactive arabinose, galactose or proline into this fraction was not stimulated by auxin. These results contradict the hypothesis that AGPs function as wall-loosening agents in auxin-mediated extension growth. The results are compatible with the notion that AGPs serve as developmental markers defining particular features of future cell differentiation. The specific association of the epitopes recognized by JIM13 and JIM14 with disintegrating cells suggests that the related AGPs identify those cells of the coleoptile which are committed to programmed cell death.

Cell Death↗

PubMed1995

A multicenter clinical trial of PerioGard in distinguishing between diseased and healthy periodontal sites. (I). Study design, methodology and therapeutic outcome.

We designed and performed a multicenter clinical trial to determine the relationship between measurements of the level of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) to other measures used to detect periodontal disease and monitor outcome of treatment, including pocket depth and gingival inflammation. 32 periodontitis patients were enrolled at the University of Washington, Seattle, 30 at the University of Florida, Gainesville, and 34 at the University of Illinois, Chicago. 10 periodontally normal control subjects were enrolled at each location. 8 diseased and 4 healthy sites were designated for study in each patient and 8 healthy sites designated in each control subject. Measures of disease included pocket depth, severity of gingival inflammation, and GCF volume. AST levels were measured using the PerioGard test kit. Clinical measurements were made and GCF samples harvested and tested 2x before and 2x after therapy consisting of scaling and root planing under local anesthetic. Specific design and other issues are discussed, including selection of patients and control subjects, sample size, selection of experimental test sites, methods for assessment of diseased and therapeutic improvement, harvesting of GCF and selection of appropriate biostatistical methods for data analysis. Demographics of the patient populations at the 3 locations are reported. As expected, therapy induced only negligible changes in the measures of disease at healthy sites in control subjects, and relatively minor improvement in healthy sites in patients. In contrast, statistically significant improvement relative to pretreatment baseline status in all 3 measures of disease was observed for diseased sites at all 3 study locations with all p-values less than 0.0002. The magnitude of improvement was comparable to that reported previously by others. The % of PerioGard-positive sites decreased significantly between the screening baseline and both post-treatment visits for patients at all 3 locations, with p values of 0.0001 to <0.0008.

Adult↗

PubMed1993

Factors associated with burnout of physical therapists in Massachusetts rehabilitation hospitals.

BACKGROUND AND PURPOSE: The purpose of this study was to determine the factors associated with burnout in physical therapists at rehabilitation hospitals. Burnout is a serious concern because it can lead to psychosomatic complaints, work-associated withdrawal behaviors, and a decreased quality of care. SUBJECTS: Full-time, nonadministrative, nonsupervisory, staff physical therapists working in inpatient settings in rehabilitation hospitals in Massachusetts were surveyed. METHODS: The survey included demographic data, the Maslach Burnout Inventory (MBI), and questions designed to address attributes of the work environment and individual personalities. The MBI scores were calculated according to instructions provided by publishers to determine the degree of burnout experienced by this group. Questions involving personality and work environment were submitted to factor analyses. Multivariate analyses were done to determine the factors related to burnout. RESULTS: Forty-six percent of the respondents scored high on the emotional exhaustion subscale of the MBI, 20% scored high on the depersonalization subscale, and 60% scored low on the personal accomplishment subscale. As a whole, the sample demonstrated moderate burnout. Three factors emerged from the factor analysis. The factors communication/connectedness, achievement, and time constraints accounted for 69% of the variability in emotional exhaustion and 73% of the variability in depersonalization and personal accomplishment. Burnout was not significantly associated with the therapists' number of years of practice, number of years on the job, or number of patients seen daily. CONCLUSION AND DISCUSSION: Recognition of factors contributing to burnout may prove to be instrumental in the prevention of burnout in physical therapists and the problems that arise from it.

Adult↗

PubMed1981

The pathology and biology of uremic pneumonitis.

Uremic pneumonitis is included in the "adult respiratory distress syndrome". Diffuse alveolo-capillary damage, interstitial and intraalveolar edema, widespread atelectases, alveolar haemorrhages and pulmonary hyaline membranes are characteristic but not pathognomonic findings in uremic pneumonitits. Investigations showed that uremic pneumonitis results from diffuse permeability disorders of the alveolo-capillary permeability combined with an intrarenal or generalized consumption of coagulation factors, and a subsequent state of hypercoagulability. A comparative morphological and clinical analysis of 66 autopsy cases with severe uremia showed that neither the intensity of the urea retention nor the intensity of the creatinine retention correlate with the morphologic symptoms of uremic pneumonitis. Uremia may induce the basic alveolocapillary damage and the subsequent plasmatic leakage into the alveoli and alveolar ducts but uremia has obviously no influence on the simultaneous formation of pulmonary hyaline membranes and intraalveolar bleedings. Hyaline membranes and intraalveolar bleedings are the entire equivalents of the immediate procoagulative activity of the underlying diseases.

Creatinine↗