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Biomedical subjects

M Geyer

Publications and source records attributed to M Geyer.

At least 37 records · Page 2Linked to original sources
PubMed2001

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity.

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Structure--function relationships in HIV-1 Nef.

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure-function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.

Amino Acid Motifs↗
PubMed2001

Analysis of ankyrin repeats reveals how a single point mutation in RFXANK results in bare lymphocyte syndrome.

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

Amino Acid Sequence↗
PubMed2001

[Central relationship patterns in comparison with different objects].

In the present study the Relationship Episode Paradigm Interviews of 70 female patients with different psychoneurotic diseases were analysed with respect to object-specific patterns with the CCRT method. The most frequent categories are the same in all relationship episodes and in subsamples of relationship episodes with mother and father. These categories are also predominant in episodes with women and men. Relationship episodes with mother do not differ from episodes with father, and relationship episodes with women are not different from episodes with men. But there are substantial differences in relationship episodes with the mother and women and between episodes with the father and men. Patients recount much more positive relationship patterns with women and men than with their parents. This could be understood as a hint of interpersonal resources.

Adult↗
PubMed2000

[Utilization of medical services and medication intake of patients over 60 in Germany--health related, social structure related, socio-demographic and subjective factors].

In a community sample of 394 elderly aged 61 years and older from East and West Germany, diseases, contacts with general practitioners and specialists, the use of medicine, attitudes regarding health and illness, the subjective health, psychic problems, social support, social integration, social burden, and socio-demographic variables were assessed. Based on these data the determinants for the contact of physicians and the use of medicine were analyzed. The results confirmed the frequency of multimorbidity in the elderly; on average we found three different diseases at the same time for each person. In nearly 10% of the sample we found seven diagnoses existing at the same time. 88% had contact with a general practitioner at least once a year, 97% had contact either with a general practitioner or with a specialist once a year. 55.8% took at least one medicine each day. The number of diseases existing at the same time was the most determining variable for the contact of physicians and the use of medicine. Furthermore, the elderly had more contact with physicians and took more medicine if they thought they were susceptible to diseases in a high degree, and if they rated their own health as poor. Fewer contacts with physicians and a lower use of medicine were found in those elderly that rated health behavior as little useful, that had low control beliefs regarding their own health, and that experienced only a low degree of health-related limitations in their everyday life. Furthermore, we found a higher use of medicine if there was little social support. There were no significant age-related or sex-related differences regarding the contact of practitioners or the use of medicine.

Aged↗
PubMed2000

[Illness-coping in a population-based representative sample. Situational, sociodemographic and social influences].

In spite of the considerable volume of current coping research there is a noticeable lack of studies based on large representative samples. In this study a sample of 2179 subjects of 16 to 96 years of age that was representative of the entire population of Germany, was asked as to how they now cope, how they have coped in the past or how they would cope with: 1. an existing illness, 2. a past illness and 3. an imagined (non-existent) illness. The instrument used was the questionnaire version of the "Berner Bewältigungsformen" (BEFO), a coping questionnaire with 28 systematically derived coping dimensions that are defined independently of the usual empirical-topographical dimensions (behavioural, cognitive and emotional) of the type of illness. The enquiry was concerned with the influence of varying situational (proximity of illness, subjective evaluation of personal health, psychological symptoms), socio-demographic (age, gender) and social (social support) variables on the choice of coping mechanisms. The results showed that each of these factors influences the repertoire of coping mechanisms to varying degrees. This systematic variability in a representative sample should be taken into account in any evaluation of coping with illness in clinical sub-groups.

Adaptation, Psychological↗
PubMed2000

Interactions between equine cyclin T1, Tat, and TAR are disrupted by a leucine-to-valine substitution found in human cyclin T1.

Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation.

Amino Acid Sequence↗
PubMed2000

p21-activated kinase 1 plays a critical role in cellular activation by Nef.

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.

Animals↗
PubMed2000

[Not Available].

It may be possible to identify elements of psychosomatic/psychotherapeutic pri-mary care in the Federal Republic of Germany and the former German Democratic Republic by comparing the current state of basic psychosomatic care (BPSC) in the eastern and western part of Berlin. 278 Berlin patients with psychosocial problems were recruited 1995 for a basic documentation system of the BPSC in connection with a project supported by the Federal Ministry for Health at the Berlin Center. Their data were compared with re-gard to diagnostic and therapeutic measures applied in the eastern and western part of Berlin. Furthermore, 617 questionnaires on basic psychosomatic care were filled out by physicians in private practice in both parts of the city in 1994. Despite the identical or lower assessment of their patients* biopsychosocial stress, East Berlin physicians take considerably more therapeutic measures than their collea-gues in West Berlin. There are only minor differences between physicians from the eastern and western part of the city despite variances in the training and advanced training systems for BPSC. The possible causes are discussed.

Diagnosis↗
PubMed2000

[Method of plan formulation: first German language rehabilitation study of the Joseph Weiss "control mastery theory"].

The paper gives a survey of the "Control Mastery Theory" (CMT) of Joseph Weiss. CMT is a cognitive-affective, psychoanalytic theory of the psychotherapeutic process. The San Francisco Psychotherapy Research Group empirically investigated und confirmed several concepts of the CMT. Some research results are summarized and the "Plan Formulation Method" is described in detail. The results of the first German reliability study applying the plan formulation method on the case "Amalie" show that the method can be used reliably for research purposes outside the USA.

Adult↗
PubMed1999

Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif.

hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.

Aluminum Compounds↗
PubMed1999

Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.

It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.

Cell Cycle Proteins↗
PubMed1999

Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae.

The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.

Amino Acid Sequence↗
PubMed1999

Structural and biochemical analysis of Ras-effector signaling via RalGDS.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.

Crystallography, X-Ray↗
PubMed1999

Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

Amino Acid Sequence↗