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L Schmitt

Publications and source records attributed to L Schmitt.

At least 37 records · Page 2Linked to original sources
PubMed2000

Affinity, specificity, diversity: a challenge for the ABC transporter TAP in cellular immunity.

The immune system is a perfect piece of machinery that serves only one purpose: to protect and defend our organism. The fact that we are still alive, although surrounded by a hostile environment, demonstrates impressively how well the immune system performs this task. Over the past decades our knowledge about the functions and components of the immune system has increased exponentially. Especially for the humoral immune system and its key players, the antibodies, a detailed knowledge about structure and function as well as regulation and communication between the individual components exists. In case of the cellular immune system that knowledge is not as profound as for the humoral counterpart. In this article we do not intend to give a complete overview about both branches of the immune system. Rather, we want to focus on an essential protein of the cellular immune system, the transporter associated with antigen processing (TAP). This transmembrane protein complex displays unique properties with respect to recognition and translocation of a vast spectrum of antigenic peptides. Its important role within the major histocompatibility complex (MHC) class I mediated immune response has been utilized by some viruses which target TAP to hide and escape from a deadly attack by the immune system. Here we summarize the existing data and analyze structural and mechanistic aspects of recognition and transport by which this transporter performs its task.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Kinetics of peptide binding to the class II MHC protein I-Ek.

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.

Amino Acid Sequence↗
PubMed2000

A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy.

In recent years, the atomic force microscope (AFM) has contributed much to our understanding of the molecular forces involved in various high-affinity receptor-ligand systems. However, a universal anchor system for such measurements is still required. This would open up new possibilities for the study of biological recognition processes and for the establishment of high-throughput screening applications. One such candidate is the N-nitrilo-triacetic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine tag acts as a high-affinity recognition site for the NTA chelator. Accordingly, we have investigated the possibility of using this approach in single-molecule force measurements. Using a histidine-peptide as a model system, we have determined the binding force for various metal ions. At a loading rate of 0.5 microm/s, the determined forces varied from 22 +/- 4 to 58 +/- 5 pN. Most importantly, no interaction was detected for Ca(2+) and Mg(2+) up to concentrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demonstrated the reversibility of the approach. Here the molecular interactions were turned off (EDTA) and on (metal reloading) in a switch-like fashion. Our results show that the NTA/His-tag system will expand the "molecular toolboxes" with which receptor-ligand systems can be investigated at the single-molecule level.

Chelating Agents↗
PubMed2000

Axial penile rigidity as primary efficacy outcome during multi-institutional in-office dose titration clinical trials with alprostadil alfadex in patients with erectile dysfunction. Alprostadil Alfadex Study Group.

The purpose of the study was to utilize axial penile buckling testing as a primary efficacy variable of erection quality during multi-institutional in-office dose titration testing with alprostadil alfadex, prostaglandin E1 (PGE1)-alpha-cyclodextrin, (EDEX /VIRIDAL, Schwarz Pharma) in patients with erectile dysfunction. In 41 study sites, in three different dose titration studies involving 894 patients with impotence >6 months, a buckling test was performed and repeated every 10 min for up to 60 min, within 30 min following alprostadil alfadex administration, or when two consecutive circumference measurements reached maximum values. The buckling device consisted of a standard weight scale attached to a 2 inch diameter plastic cap with a concavity on its ventral surface (H. Eric Richards, Inc., Canton MA). A positive test was associated with absent penile shaft buckling to a downward force of 1.0 kg, slowly applied to the glans in the axis of the erect shaft. A total of 630 (71%) patients experienced at least one positive buckling test. Three consecutive positive buckling tests, implying a functionally rigid erection for at least 20 min, were noted in 521 (58%) patients. There were high correlations between the presence of three consecutive positive buckling tests following alprostadil alfadex injection and the patient's (83% of cases) and the investigator's (88% of cases) evaluation of adequacy of erection for intercourse. Similarly, there were high correlations between the presence of negative buckling tests and the patient's (95% of cases) and the investigator's (96% of cases) evaluation of inadequacy of erection for intercourse. The axial penile buckling test offers a simple, reliable, and inexpensive method to objectively quantify erectile response following in-office dose titration of intracavernosal alprostadil alfadex. The high correlation to subjective patient/investigator assessment adds to the validity of the test.

Adult↗
PubMed2000

[Trauma, traumatic stress and depression following an airline catastrophy].

OBJECTIVES: Acute stress responses following a trauma indicate a sensitivity to posttraumatic stress disorder (PTSD), and is often comorbid with depression. Earlier exposure to a traumatic event can be an additional risk factor in PTSD development. METHOD: Eight injured patients hospitalized after a major air disaster were monitored and assessed for a month. The symptoms of acute stress response (ASR), PTSD, and depression were assessed using DSM-IV criteria immediately following the accident, then each week thereafter. The Impact of Event Scale (IES) was completed on the 30th day (D30). RESULTS: Four patients presented with an ASR, and 3 of them had a PTSD at D30. Of those 3 patients with PTSD, 2 presented with an associated depression. These 2 patients had been exposed to a traumatic event before the disaster; and a significant relation was found between the history of the earlier trauma and the PTSD associated with depression. CONCLUSION: The traumatized victims with a history of earlier traumas seem more susceptible to developing a PTSD associated with depression.

Accidents, Aviation↗
PubMed1999

Catalysis of peptide dissociation from class II MHC-peptide complexes.

Certain peptides such as dynorphin A [dynA-(1-13)] enhance the release of antigenic peptides bound to class II MHC molecules at neutral pH. This enhanced release has been termed push off. Previous work has shown that the antigenic pigeon cytochrome c peptide PCC-(89-104) has at least two conformational isomers when bound to the class II MHC protein I-Ek. We have accordingly studied the push off of PCC-(89-104) from the complex PCC-(89-104)/I-Ek to see whether these isomeric conformations are distinguished by the push-off effect. A comparison of the association and dissociation kinetics of PCC-(89-104)/I-Ek in the presence of dynA-(1-13) shows that dynA-(1-13) does not simply replace PCC-(89-104) but rather acts catalytically. The major product is peptide-free I-Ek, which is receptive to further peptide binding. Evidence is presented that a two peptide-one MHC complex is formed in solution. This ternary complex represents the first step of the mechanism of push off. 19F NMR data are presented that indicate that dynA-(1-13) interacts specifically with only one of the two isomeric complexes of PCC-(89-104) and I-Ek. A push-off mechanism is proposed in which dynA-(1-13) binds outside the peptide binding groove. In a second step, the dissociation of one of the two isomers is specifically enhanced. Thus the push-off effect may be useful for identifying conformational isomers and for separating them.

Animals↗
PubMed1999

Conformational isomers of a class II MHC-peptide complex in solution.

A number of kinetic measurements of peptide dissociation from class II MHC-peptide complexes provide compelling evidence for the existence of conformational isomers in solution. There is evidence that T-lymphocytes can distinguish such isomers. However, virtually nothing is known about the structure of these isomers. Accordingly, we have investigated a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c residues 89-104 (PCC) by 19F-NMR. Two fluorine labels were placed on the PCC peptide; one fluorine label was placed at a MHC contact site, the other at a position involved in T-cell receptor (TCR) recognition. Introduction of these labels did not alter the observed kinetics of the PCC/I-Ek complex. The NMR data show two conformational isomers of this immunogenic complex. The presence of conformational isomers at a TCR contact site suggests that these structures may be recognized differently by the TCR. The agreement between the dissociation kinetics and the 19F-NMR data demonstrate that kinetic heterogeneity is correlated with structural counterparts observed by NMR. Dissociations in the presence of dimethyl sulfoxide were used to show that the rate of interconversion of these conformational isomers at pH 7.0 is low, with a lifetime on the order of hours or more. Modification of a peptide residue of PCC occupying the minor MHC binding pocket P6 alters the 19F-NMR spectra of both labels. This demonstrates that distant changes of amino acid residues can influence the conformation of the whole antigenic peptide inside the MHC binding cleft.

Animals↗
PubMed1999

Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study.

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

DNA, Single-Stranded↗
PubMed1999

[Acute peri-traumatic dissociative experiences: assessment and course].

Previous research has identified acute stress symptoms, particularly peri-traumatic dissociative symptoms (the distortion of consciousness, depersonalization, derealization, automatic movements, flashbacks with illusions or hallucinations), as risk factors for the development of later posttraumatic stress disorder. Numerous retrospective assessments and current prospective studies confirm these findings. It is suggested that peri-traumatic dissociation be assessed immediately after traumatic exposure and during the weeks following. But traumatized victims may present other categories of acute reactions; panic attacks, acute depression, conversion reaction, excessive emotional expression, and psychotic reactions. Brief reactive psychosis is a major differential diagnosis with peri-traumatic dissociative experiences. During emergency interventions it may be difficult to distinguish between dissociative and psychotic symptoms. It is cautioned that these disorders be evaluated with a follow-up of several months.

Depression↗
PubMed1998

Kinetic isomers of a class II MHC-peptide complex.

Class II major histocompatibility (MHC) molecules bind fragments of antigens and present them to T cells. The triggering of the T-cell receptor (TCR) of CD4(+) T-helper cells by these protein-peptide complexes is a key event in the generation of a cellular immune response. In the context of this interaction, it is generally assumed that class II MHC-peptide complexes adopt a single recognition structure at the cell surface. On the other hand, kinetic analysis has revealed that a number of class II MHC-peptide complexes show biphasic dissociation kinetics, indicating the presence of multiple kinetic isomers. Here, we demonstrate that a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c (PCC) displays monophasic as well as biphasic dissociation kinetics. While a simple monophasic dissociation curve was obtained at neutral pH, the complex showed biphasic dissociation behavior at acidic pH. This shift was independent of the ionic strength of the solution. Moreover, the short-lived isomer could be regenerated from a pool of kinetically homogeneous long-lived complexes. This demonstrates that the isomers interconvert and exist in a pH-sensitive equilibrium. Altering the peptide residue of PCC that occupies the P6 pocket of I-Ek results in a class II MHC-peptide complex that shows only monophasic dissociation, indicating that the glutamine at this position plays a key role in the kinetic heterogeneity of the complex.

Amino Acid Sequence↗
PubMed1998

Paralysis of B7 co-stimulation through the effect of viral IL-10 on T cells as a mechanism of local tolerance induction.

The Epstein-Barr virus (EBV) encodes an open reading frame with significant homology to the cellular IL-10 gene. This viral IL-10 (vIL-10) might enable EBV to evade antiviral T cells. We employed transfectants of a murine tumor cell line (P815) to investigate whether vIL-10 interferes with the first (antigenic) or second (co-stimulatory) signal of T cell activation. Untransfected P815 cells caused tumors in syngeneic DBA/2 mice after s.c. inoculation. In contrast, transfectants that provided either a strong antigenic stimulus (P815-Kb cells) or a strong co-stimulatory signal (P815-B7 cells) were rejected. Injection of double-transfected P815 cells expressing Kb and secreting high levels of vIL-10 (P815-Kb-vIL-10) did not result in tumor growth. We then investigated whether vIL-10 could paralyse co-stimulation by B7 under the same conditions. Therefore P815-B7 cells were mixed with vIL-10-secreting P815-Kb cells and co-injected into DBA/2 animals. Most of these mice developed a tumor. Explanted tumor cells expressed the B7 molecule but not the Kb antigen. These observations in vivo were mirrored by experiments in vitro: vIL-10 could induce T cell tolerance towards P815-B7 cells but not P815-Kb cells. Taken together our results suggest that vIL-10 acts directly on T cells to inhibit co-stimulatory signals mediated via B7 receptors such as CD28 or CTLA-4.

Animals↗
PubMed1997

A comparative study of the effects of yohimbine and anetholtrithione on salivary secretion in depressed patients treated with psychotropic drugs.

OBJECTIVE: The present study compares the effects of yohimbine, an alpha 2 adrenoceptor antagonist, and anetholtrithione, a reference drug in the treatment of dry mouth, in patients treated with psychotropic drugs (tricyclic antidepressants or neuroleptics) and suffering from xerostomia. METHODS: Ten patients were included in a randomized, double-blind, cross-over study, and receiving after yohimbine (3 x 6 mg per day) or anetholtrithione (3 x 25 mg per day) orally for 5 days. Salivary secretion was estimated under resting conditions, before any drug, and then on day 6, 1 h after the ingestion of yohimbine or anetholtrithione. RESULTS: Compared with basal secretion, the increase in salivary flow was significantly more marked after yohimbine than after anetholtrithione. CONCLUSION: This study demonstrates the sialogenic effect of yohimbine in drug-induced dry mouth.

Adrenergic alpha-Antagonists↗
PubMed1997

[Main syndromes revealed in a consultation specially focusing on memory].

This paper reports our 2-year experience in a memory clinic practice at a University Hospital. During these 2 years, 128 patients with memory complaints were evaluated by a neurologist, a psychiatrist, a geriatrist and a neuropsychologist using a standardized test battery; only 58 of these patients actually presented with objective memory deficits. These memory impairments were mainly observed in patients with neurologic disorders (principally dementia) and in patients with psychiatric disorders (principally depression, major anxiety and psychiatric diseases with troubles of personality). The aim of this memory clinic is to identify the origin of memory disorders, provide a pharmacological treatment when required, propose appropriate assessment and follow up, and diagnose early symptoms of dementia.

Adult↗