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J Benz

Publications and source records attributed to J Benz.

At least 19 recordsLinked to original sources
PubMed1999

Interaction of translation initiation factor eIF4G with eIF4A in the yeast Saccharomyces cerevisiae.

Eukaryotic initiation factor (eIF) 4A is an essential protein that, in conjunction with eIF4B, catalyzes the ATP-dependent melting of RNA secondary structure in the 5'-untranslated region of mRNA during translation initiation. In higher eukaryotes, eIF4A is assumed to be recruited to the mRNA through its interaction with eIF4G. However, the failure to detect this interaction in yeast brought into question the generality of this model. The work presented here demonstrates that yeast eIF4G interacts with eIF4A both in vivo and in vitro. The eIF4A-binding site was mapped to amino acids 542-883 of yeast eIF4G1. Expression in yeast cells of the eIF4G1 domain that binds eIF4A results in cell growth inhibition, and addition of this domain to an eIF4A-dependent in vitro system inhibits translation in a dose-dependent manner. Both in vitro translation and cell growth can be specifically restored by increasing the eIF4A concentration. These data demonstrate that yeast eIF4A and eIF4G interact and suggest that this interaction is required for translation and cell growth.

Eukaryotic Initiation Factor-4A↗
PubMed1999

Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family.

BACKGROUND: Translation initiation factor 4A (elF4A) is the prototype of the DEAD-box family of proteins. DEAD-box proteins are involved in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. Energy from ATP hydrolysis is used to perform RNA unwinding during initiation of mRNA translation. The presence of elF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. RESULTS: We present here the crystal structure of the nucleotide-binding domain of elF4A at 2.0 A and the structures with bound adenosinediphosphate and adenosinetriphosphate at 2.2 A and 2.4 A resolution, respectively. The structure of the apo form of the enzyme has been determined by multiple isomorphous replacement. The ATPase domain contains a central seven-stranded beta sheet flanked by nine alpha helices. Despite low sequence homology to the NTPase domains of RNA and DNA helicases, the three-dimensional fold of elF4A is nearly identical to the DNA helicase PcrA of Bacillus stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. CONCLUSIONS: We have determined the crystal structure of the N-terminal domain of the elF4A from yeast as the first structure of a member of the DEAD-box protein family. The complex of the protein with bound ADP and ATP offers insight into the mechanism of ATP hydrolysis and the transfer of energy to unwind RNA. The identical fold of the ATPase domain of the DNA helicase PcrA of B. stearothermophilus and the RNA helicase of hepatitis C virus suggests a common fold for all ATPase domains of DExx- and DEAD-box proteins.

Adenosine Diphosphate↗
PubMed1999

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

Adenosine Monophosphate↗
PubMed1998

Interactions of benzodiazepine derivatives with annexins.

Human annexins III and V, members of the annexin family of calcium- and membrane-binding proteins, were complexed within the crystals with BDA452, a new 1,4-benzodiazepine derivative by soaking and co-crystallization methods. The crystal structures of the complexes were analyzed by x-ray crystallography and refined to 2.3- and 3.0-A resolution. BDA452 binds to a cleft which is located close to the N-terminus opposite to the membrane binding side of the proteins. Biophysical studies of the interactions of various benzodiazepine derivatives with annexins were performed to analyze the binding of benzodiazepines to annexins and their effects on the annexin-induced calcium influx into phosphatidylserine/phosphatidylethanolamine liposomes. Different effects were observed with a variety of benzodiazepines and different annexins depending on both the ligand and the protein. Almost opposite effects on annexin function are elicited by BDA250 and diazepam, its 7-chloro-derivative. We conclude that benzodiazepines modulate the calcium influx activity of annexins allosterically by stabilizing or destabilizing the conducting state of peripherally bound annexins in agreement with suggestions by Kaneko (Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997) J. Mol. Biol., in press).

Amino Acid Sequence↗
PubMed1997

Voltage dependent binding of annexin V, annexin VI and annexin VII-core to acidic phospholipid membranes.

Annexin V, VI and VII-core (delta1-107) are members of the annexin protein family and bind to acidic phospholipid membranes in a calcium dependent manner. They also show ion channel activity under certain conditions. As annexins bind peripherally to lipid membranes, ion channel formation must consist of at least two steps: An adsorption reaction regulating the binding of annexin to the membrane surface and the opening and closing of the active species controlling the channel activity. By using the baseline current through the patch clamp seal as a probe for unoccupied binding sites at the membrane, we show that the adsorption of annexins to membranes is not only calcium dependent but also strongly voltage dependent. Whereas the free transfer energies at low calcium concentrations are similar for all three annexins, the binding of annexin V becomes much tighter with higher calcium levels, compared to annexin VI and VII-core. This correlates with the finding that annexin VI and VII-core display channel activity much more often than annexin V if one assumes that a high coverage of the membrane surface with annexins stabilizes the bilayer. At higher protein concentrations weaker binding is observed in agreement with the previously reported anti-cooperativity of membrane binding.

Adsorption↗
PubMed1997

Crystal structure of the C-terminal tetrad repeat from synexin (annexin VII) of Dictyostelium discoideum.

Synexin (annexin VII) is a cytosolic Ca(2+)-binding protein that promotes membrane fusion and forms voltage-regulated ion channels in artificial and natural membranes. The crystal structure of the C-terminal tetrad repeat from recombinant synexin (annexin VII) of Dictyostelium discoideum was solved to 2.45 A resolution. The protein crystallized in a dimeric form with two molecules joined face-to-face by their convex sides. Mainly hydrogen bonds and van der Waals contacts are involved in dimer formation, while not Ca2+ is bound to the conserved Ca(2+)-binding sites. The truncated N terminus is folded into a short antiparallel beta-sheet, from which the side-chain of Tyr111 penetrates sideways into the central, hydrophilic pore and may directly affect the ion channel activity. In order to investigate the structure of the missing N-terminal domain, we synthesized a 37-membered peptide of the N-terminal tail, (GYPPQQ)6G. CD and NMR studies showed a random coil conformation of the peptide in solution, suggesting for the synexin N terminus the lack of a well-ordered, three-dimensional fold.

Amino Acid Sequence↗
PubMed1997

Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction.

Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t1/2 = 54.7 degrees C (heating rate of 2.34 K min-1), delta H0 = 690 kJ mol-1, and delta Cp = 10.3 kJ mol-1 K-1 which correspondends to a value of delta G0D (20 degrees C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration C1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the delta Gapp versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature delta t1/2 (= 18 degrees C) when the GdnHCl concentration is increased from 0 to 1 M. "Regular" proteins experience an average decrease in the transition temperature of 8 +/- 2 degrees C per 1 M change in the concentration of GdnHCl.

Annexin A5↗
PubMed1997

Valsartan, a new angiotensin II receptor antagonist: a double-blind study comparing the incidence of cough with lisinopril and hydrochlorothiazide.

The present study compares the occurrence of a dry, persistent cough with doses of 80 mg of valsartan, 10 mg of lisinopril, or 25 mg of hydrochlorothiazide in patients with a history of angiotensin-converting enzyme inhibitor-induced cough. This was a randomized, double-blind, active-controlled, parallel group, multicenter trial involving 129 adult outpatients with essential hypertension. After confirmation of angiotensin-converting enzyme inhibitor-induced cough during a 2 to 4 week challenge with lisinopril (followed by a washout period of 2 weeks), patients were randomized to receive 6 weeks of double-blind treatment once daily with 80 mg valsartan, 10 mg lisinopril, or 25 mg hydrochlorothiazide. Assessments were made at baseline and after 3 and 6 weeks of treatment. Comparability of response to treatment was assessed by mean sitting diastolic and systolic blood pressure at the end of treatment. The occurrence of a dry, persistent cough was significantly less (P < 0.001) at 3 and 6 weeks with valsartan (19.5%) than with lisinopril (68.9%), with no significant difference between valsartan and hydrochlorothiazide (19.0%). There were no statistically significant differences in reduction of blood pressure among the three treatment groups. The overall incidence of adverse experiences, whether or not treatment-related, was highest for lisinopril (86.7%) compared with valsartan (57.1%), and hydrochlorothiazide (61.9%). A dry cough in the lisinopril group accounted for this difference. There were no clinically significant changes in physical signs or in results of clinical laboratory evaluations during double-blind treatment, except for from metabolic changes in 3 patients receiving hydrochlorothiazide. In hypertensive patients with a history of angiotensin-converting enzyme inhibitor-induced cough, a single daily dose of 80 mg of valsartan produced therapeutic efficacy comparable to lisinopril but with significantly less cough.

Angiotensin Receptor Antagonists↗
PubMed1996

The structure of recombinant human annexin VI in crystals and membrane-bound.

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.

Annexin A6↗
PubMed1996

Structural and functional characterisation of the voltage sensor in the ion channel human annexin V.

The ion channel properties of human annexin V, a calcium- and phospholipid-binding protein of the annexin family, have been structurally and functionally investigated by analysing the mutant Glu112 -->Gly. Glu112 forms a salt bridge with Arg271 located in the interior of the hydrophilic pore of the molecule which is conserved within the annexin family. The crystal structures of the mutant and wild-type proteins are very similar and show only marginal conformational changes around the mutation site. Electron microscopic images show a conserved four-domain structure upon membrane binding as in the wild-type annexin V. The channel properties of the mutant are drastically changed, as the mutant has lost the voltage-dependent channel gating and the selectivity for calcium ions over monovalent cations. These results strongly support the hypothesis that the central, hydrophilic pore is the ion-conducting pathway.

Annexin A5↗
PubMed1996

The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein.

Annexin II binds in a calcium-dependent manner to acidic phospholipids and is a substrate of some protein kinases. An N-terminally shortened form of human annexin II was crystallized and its molecular structure determined. It is very similar to two previously described members of this protein family, annexin I and annexin V. The protein structure is nearly completely alpha-helical organized as four compact domains which consist of five alpha-helices each. The domains surround a hydrophilic pore. The calcium binding sites are located at the convex side of the structure as in annexin V. Recombinant and natural porcine annexin II are active as ion channel with characteristics similar to annexin V, while N-terminally shortened annexin II and the heterotetramer (annexin II-p11)2 are inactive. Two cysteine residues, Cys133 and Cys262, form a disulphide bridge connecting domains II and III, adding further weight to the notion that ion channel activity does not require major structural rearrangements.

Amino Acid Sequence↗
PubMed1994

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

Annexin A5↗
PubMed1993

[Epidemiology of antirheumatic drug-induced ulcer in comparison with peptic gastroduodenal ulcer. Five-year analysis of a hospital patient sample].

Within the framework of a five-year analysis of a hospital population (n = 503), the epidemiological and diagnostic data of patients with non-steroidal anti-inflammatory drugs-associated ulcers of the stomach and duodenum (AIU) were evaluated and compared with those of patients with peptic ulcers (PUC) in the stomach and duodenum with no history of NSAIDs use. With respect to age and sex, patients with AIU were significantly older, in particular in the case of gastric ulcer, and revealed a predominance of female sex as compared with those with PU. Episodes of ulcer disease showed no seasonal dependence, either in the AIU or in the PU group. The use of tobacco and alcohol was comparable in both groups. Nor was any difference found between city and rural populations in either group. Laboratory data revealed a higher rate of pathological deviations in the AIU as compared with the PU group, which, however, was unequivocally due to the larger percentage of elderly patients in the former. With respect to blood group distribution, no differences were observed between the groups. An analysis of ulcer site showed a small preponderance of gastric ulcer (59%) over duodenal ulcer (41%) in the AIU group, while the distribution in the case of the PU group was virtually identical (gastric ulcer 51%, duodenal ulcer 49%), with only small differences in site within the stomach and duodenal bulb resp. Analysis of the risk indicators for ulcer bleeding (AIU 66%, PU 50%) revealed the following pattern: in the case of AIU, patients aged over 65, and in the case of AIU and PU, overweight males, bled significantly more often than, respectively, younger patients and female or normal-weight patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Humoral and cellular immunity in HIV positive and HIV negative Helicobacter pylori infected patients.

The prevalence of H. pylori associated gastritis seems to be different in HIV positive and HIV negative patients. Therefore a correlation to immunodeficiency can be postulated. The histology of gastritis, status of H. pylori infection and parameters of humoral and cellular immune response were investigated in 41 HIV positive and 47 HIV negative patients, who were subjected to upper endoscopy for the evaluation of gastrointestinal symptoms. In HIV positive patients 37% had active chronic gastritis against 62% of the HIV negative patients. In 73% of HIV positive cases of active chronic gastritis H. pylori was detected by bacteriological culture and/or Warthin-Starry stain. In HIV negative patients active chronic gastritis was always associated to H. pylori infection. Production of antibodies as measured by two commercially available ELISA tests was significant in HIV positive and HIV negative patients; both tests correlated well with H. pylori detection by culture or direct microscopy. Immunoglobulin class specific immunoblots corresponded to the ELISA results in HIV negative patients but to a lesser extent in the HIV positive group which was assumed to be related to unspecific polyclonal activation in these patients. Systemic cellular immunity was investigated by proliferation assays of peripheral blood mononuclear cells (PBMC). Proliferative response to the unspecific mitogen PHA was reduced in HIV positive patients. A sonicated H. pylori antigen failed to induce lymphocyte proliferation. The antimitogenic effect was also seen in case of coincubation with PHA. This observation was independent of H. pylori and HIV infection status. We conclude that in HIV positive as in HIV negative patients active chronic gastritis is predominantly related to H. pylori infection. The prevalence of H. pylori associated gastritis in HIV positive patients is significantly reduced (p < 0.025) compared to HIV negative controls. Decreased susceptibility to H. pylori infection in HIV positive patients may not be explained by the abnormal reactivity of their humoral or cellular immune response.

Adult↗
PubMed1992

[The effectiveness of preventive use of antibiotics in Cesarean section with a single dose: comparison of cefotaxime with amoxicillin plus clavulanic acid].

The effectiveness of a single prophylactic dose of two broad spectrum antibiotics, during Caesarean section, was compared and evaluated. In 119 consecutive cases at the Cantonal Hospital in Winterthur, Switzerland, either 1 g i.v. cefotaxime or 1,2 g amoxicillin plus clavulanic acid were administered after clamping the umbilical cord during Caesarean section. The study shows that single dose prophylaxis of either regimen provides adequate cover in preventing morbidity in Caesarean section, in both high-risk and low-risk groups.

Adult↗
PubMed1992

[Effectiveness of estriol depot vaginal suppositories in postmenopausal women with urogenital climacteric manifestations].

In this study, 43 postmenopausal patients with urogenital complaints such as vaginal dryness, incontinence, or bleeding with intercourse were treated with weekly applications of Orthogynest vaginal estriol suppositories in a new depot form. A marked increase in the vaginal epithelium (A. Schmitt score) and a significant decrease in symptoms due to vaginal atrophy were noted. The depot form allows for decreased dosing frequency, leading to improved compliance. Systemic symptoms, such as hot flashes and emotional lability, were also noted to respond to the Orthogynest suppositories.

Administration, Intravaginal↗