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Biomedical subjects

R Frank

Publications and source records attributed to R Frank.

At least 91 records · Page 5Linked to original sources
PubMed1999

Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.

Fast N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and occurs by a 'ball-and-chain'-type mechanism. In this mechanism an N-terminal protein domain (inactivation gate) occludes the pore from the cytoplasmic side. In Kv3.4 channels, inactivation is not fixed but is dynamically regulated by protein phosphorylation. Phosphorylation of several identified serine residues on the inactivation gate leads to reduction or removal of fast inactivation. Here, we investigate the structure-function basis of this phospho-regulation with nuclear magnetic resonance (NMR) spectroscopy and patch-clamp recordings using synthetic inactivation domains (ID). The dephosphorylated ID exhibited compact structure and displayed high-affinity binding to its receptor. Phosphorylation of serine residues in the N- or C-terminal half of the ID resulted in a loss of overall structural stability. However, depending on the residue(s) phosphorylated, distinct structural elements remained stable. These structural changes correlate with the distinct changes in binding and unbinding kinetics underlying the reduced inactivation potency of phosphorylated IDs.

Animals↗
PubMed1999

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase.

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.

Bacillus subtilis↗
PubMed1999

Selective formation of Gsalpha-MHC I complexes after desensitization of human platelets with iloprost.

Prolonged treatment of human platelets with the adenylate cyclase-stimulating prostacyclin analog iloprost leads to reduction in cAMP formation. Previous studies have demonstrated that this may be ascribed to modification of both receptor and Gsalpha function rather than of the catalytic component of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur. J. Biochem. 210, 539-544]. Iloprost-induced desensitization was accompanied by the formation of a Gsalpha-containing 90-kDa product in membranes treated with the bifunctional cross-linker 1,6-bismaleimidohexane. The cAMP-inducing prostanoid PGD2, which does not promote desensitization, did not cause formation of the 90-kDa species either. The long-term effect of the common G-protein activator [AlF4]- on human platelet adenylate cyclase was shown in many respects to be comparable with that of iloprost. However, [AlF4]- treatment also failed to induce the 90-kDa species, showing that different mechanisms of desensitization were operating. Treatment of the cross-linked 90-kDa complex with PNGase F demonstrated the glycoprotein nature of the Gsalpha-associated component. The 90-kDa cross-linked product was purified by consecutive immunoaffinity chromatography and preparative PAGE to apparent homogeneity. Analysis of the purified protein by MS suggested that, besides Gsalpha, the heavy chain of MHC I (HLA-A2) was part of the complex. This was confirmed by coprecipitation of Gsalpha by the monoclonal anti-(MHC I) antibody W6/32.

Adenylyl Cyclases↗
PubMed1999

Phosphorylation of components of the ER translocation site.

In many eukaryotic cells, protein secretion is regulated by extracellular signalling molecules giving rise to increased intracellular Ca2+ and activation of kinases and phosphatases. To test whether components involved in the first step of secretion, the translocation of proteins across the endoplasmic reticulum (ER) membrane, are regulated by Ca2+-dependent phosphorylation and dephosphorylation, we have investigated the effect of Ca2+ on kinases associated with the rough ER. Using purified rough microsomes from dog pancreas we found that Ca2+-dependent isoforms of protein kinase C (PKC) are associated with the rough ER and phosphorylate essential components of the protein translocation machinery. Phosphorylation of microsomal proteins by PKCs increased protein translocation efficiency in vitro. We also found that proteins of the translocation machinery became phosphorylated in intact cells. This suggests a further level of regulation of protein translocation across the ER membrane.

Animals↗
PubMed1999

The disabled 1 phosphotyrosine-binding domain binds to the internalization signals of transmembrane glycoproteins and to phospholipids.

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

Amino Acid Sequence↗
PubMed1999

Arrhythmogenic right ventricular dysplasia.

Arrhythmogenic right ventricular dysplasia (ARVD) is a new form of cardiomyopathy probably more frequent than commonly reported. It is a rare but important cause of sudden arrhythmic death in young, otherwise healthy persons, as well as a subtle cause of congestive heart failure. It may lead to temporary incapacitation with catastrophic consequences. Proper electrocardiographic criteria, echocardiography, nuclear medicine, or magnetic resonance imaging could identify most of these individuals. With the exception of full-thickness histological examination of the right ventricular free wall, contrast ventriculography remains the most definitive standard for a positive diagnosis. The wide clinical spectrum of arrhythmogenic right ventricular cardiomyopathies/dysplasia appears to be the result of one or possibly two factors: (a) replacement of most of the right ventricular myocardium by fat and (b) genetic susceptibility to environmental agents (myocarditis). Current treatment modalities include drug therapy, catheter or surgical ablative techniques, and modern treatments of congestive heart failure. Heart transplant is exceptional. Implantable defibrillators, used alone or in combination with drug therapy, will probably play an increasing role in ARVD and related cardiomyopathies.

Adipose Tissue↗
PubMed1999

Cost-effectiveness of assertive community treatment for homeless persons with severe mental illness.

BACKGROUND: Homelessness is a major public health problem among persons with severe mental illness (SMI). Cost-effective programmes that address this problem are needed. AIMS: To evaluate the cost-effectiveness of an assertive community treatment (ACT) programme for these persons in Baltimore, Maryland. METHODS: A total of 152 homeless persons with SMI were randomly allocated to either ACT or usual services. Direct treatment costs and effectiveness, represented by days of stable housing, were assessed. RESULTS: Compared with usual care, ACT costs were significantly lower for mental health in-patient days and mental health emergency room care, and significantly higher for mental health out-patient visits and treatment for substance misuse. ACT patients spent 31% more days in stable housing than those receiving usual care. ACT and usual services incurred $242 and $415 respectively in direct treatment costs per day of stable housing, an efficiency ratio of 0.58 in favour of ACT. Patterns of care and costs varied according to race. CONCLUSION: ACT provides a cost-effective approach to reducing homelessness among persons with severe and persistent mental illnesses.

Adult↗
PubMed1999

Ifosfamide, carboplatin, and etoposide: a highly effective cytoreduction and peripheral-blood progenitor-cell mobilization regimen for transplant-eligible patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate a chemotherapy regimen that consisted of ifosfamide administered as an infusion with bolus carboplatin, and etoposide (ICE) supported by granulocyte colony-stimulating factor (G-CSF) for cytoreduction and stem-cell mobilization in transplant-eligible patients with primary refractory or relapsed non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: One hundred sixty-three transplant-eligible patients with relapsed or primary refractory NHL were treated from October 1993 to December 1997 with ICE chemotherapy at Memorial Sloan-Kettering Cancer Center. Administration of three cycles of ICE chemotherapy was planned at 2-week intervals. Peripheral-blood progenitor cells were collected after cycle 3, and all patients who achieved a partial response (PR) or complete response (CR) to ICE chemotherapy were eligible to proceed to transplantation. Event-free and overall survival, ICE-related toxicity, and the number of CD34(+) cells collected after treatment with ICE and G-CSF were evaluated. RESULTS: All 163 patients were assessable for response, and there was no treatment-related mortality. A major response (CR/PR) was evident in 108 patients (66.3%); 89% of the responding patients underwent successful transplantation. Patient who underwent transplantation and achieved a CR to ICE had a superior overall survival to that of patients who achieved a PR (65% v 30%; P =.003). The median number of CD34(+) cells/kg collected was 8.4 x 10(6). The dose-limiting toxicity of ICE was hematologic, with 29.4% of patients developing grade 3/4 thrombocytopenia. There were minimal nonhematologic side effects. CONCLUSION: ICE chemotherapy, with ifosfamide administered as a 24-hour infusion to decrease CNS side effects, and the substitution of carboplatin for cisplatin to minimize nephrotoxicity, is a very effective cytoreduction and mobilization regimen in patients with NHL. Furthermore, the quality of the clinical response to ICE predicts for posttransplant outcome.

Adolescent↗
PubMed1999

An expansion of the peer-tutoring paradigm: cross-age peer tutoring of social skills among socially rejected boys.

We examined the effects of a cross-age peer-tutoring program on the social skills of 2 sixth-grade and 2 kindergarten socially rejected and isolated boys. Peer tutoring consisted of the older boys conducting social skills training with their younger tutees. The frequency of positive social interactions increased for all 4 boys, with maintenance of treatment gains following a 5-week interval.

Child↗
PubMed1999

Fat in the heart. A feature unique to the human species? Observational reflections on an unsolved problem.

Fat that is well demarcated from underlying muscle is found on the right ventricular free wall and around epicardial coronary vessels. Fat is not present in the left ventricle in normal subjects. In right ventricular dysplasia, fat and fibrosis may massively displace right ventricular myocardial tissue. It is frequently associated with some clusters of fat and fibrosis in the left ventricle. Adipocytes may be also found within fibrous tissue. In this situation it may be associated with inflammatory cellular infiltrates in both ventricles and this is called metaplastic fat. All these findings may be seen and sometimes are associated to a variable degree in the same myocardial specimen. However, fat may be interspersed with right ventricular myocardial fibres but without fibrosis or signs of inflammation. This situation is observed in more than half of the normal hearts and represents an over-looked pathologic condition only observed in the human species. The term "fat dissociation syndrome" is proposed to identify this condition. This new understanding of right ventricular myocardial structure which may be investigated by MRI may have important clinical consequences.

Adipose Tissue↗
PubMed1998

Antagonist peptide selects thymocytes expressing a class II major histocompatibility complex-restricted T cell receptor into the CD8 lineage.

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4(+) cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8(+) cells in thymus organ culture from mice impaired in CD4(+) cell development (invariant chain-deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.

Animals↗
PubMed1998

"Cut and combine": an easy membrane-supported combinatorial synthesis technique.

A combinatorial synthesis process involving sequential cycles of cutting a membrane support into pieces and combining these into groups and subjecting the groups to simultaneous solid-phase chemical reactions is demonstrated by the rapid assembly of four hundred N-terminally biotinylated, soluble, octameric peptide pools. Index patterns printed onto the synthesis membrane allowed a direct identification of the compounds. These were used to study protein kinase substrate selection in a parallel microplate adapted 32P-phosphorylation assay with subsequent spotting on a biotin-capture membrane.

Amino Acid Sequence↗
PubMed1998

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

Amino Acid Sequence↗
PubMed1998

Peroxisome biogenesis: involvement of ARF and coatomer.

Peroxisomal membrane protein (Pmp)26p (RnPex11p), a major constituent of induced rat liver peroxisomal membrane, was found to contain a COOH-terminal, cytoplasmically exposed consensus dilysine motif with the potential to bind coatomer. Biochemical as well as immunocytochemical evidence is presented showing that peroxisomes incubated with preparations of bovine brain or rat liver cytosol recruit ADP-ribosylation factor (ARF) and coatomer in a strictly guanosine 5'-O-(3-thiotriphosphate)-dependent manner. Consistent with this observation, ldlF cells expressing a temperature-sensitive mutant version of the epsilon-subunit of coatomer exhibit elongated tubular peroxisomes possibly due to impaired vesiculation at the nonpermissive temperature. Since overexpression of Pex11p in Chinese hamster ovary wild-type cells causes proliferation of peroxisomes, these data suggest that Pex11p plays an important role in peroxisome biogenesis by supporting ARF- and coatomer-dependent vesiculation of the organelles.

ADP-Ribosylation Factors↗
PubMed1998

Site-directed C3a receptor antibodies from phage display libraries.

Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.

Amino Acid Sequence↗
PubMed1998

Structure of the inactivating gate from the Shaker voltage gated K+ channel analyzed by NMR spectroscopy.

Rapid inactivation of voltage-gated K+ (Kv) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various Kv channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different Kv channels with hardly any sequence homology mediate quite similar effects even on unrelated Kv channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution.

Amino Acid Sequence↗