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J Engels

Publications and source records attributed to J Engels.

At least 19 recordsLinked to original sources
PubMed1997

Effect of preformed correct tertiary interactions on rapid two-state tendamistat folding: evidence for hairpins as initiation sites for beta-sheet formation.

The role of preformed correct side chain interactions, such as disulfide bonds, on protein folding kinetics is still not well understood. We investigated the effect of disulfide bond replacements on folding and stability of the small beta-sheet protein tendamistat. Tendamistat folds very fast (tau = 10 ms at pH 7 in water) and without detectable intermediates, which facilitates molecular interpretation of the kinetic data. Tendamistat contains two disulfide bonds, one between cysteines 11 and 27, which connects the ends of a beta-hairpin, and a second one between cysteines 45 and 73, which brings together the two outer strands of a three-stranded beta-sheet. Two single-disulfide variants of the protein were prepared by site-directed mutagenesis (tendamistat C11A/C27S and tendamistat C45A/C73A), and the effects on stability and on folding were monitored. Replacement of either disulfide bond leads to a large decrease in protein stability (DeltaDeltaG0 = 6.0 kcal/mol for the C11A/C27S variant and 5.1 kcal/mol for the C45A/C73A variant). This effect is caused both by entropic stabilization of the unfolded state and by enthalpic destabilization of the native structure. Kinetic experiments show that the main effect of fixed side chain contacts is on the unfolding rate. For both single-disulfide variants, unfolding is strongly accelerated (4250 times in the C11A/C27S variant and 250 times in the C45A/C73A variant) whereas the refolding rate constants are only slightly decreased. The activation parameters show that the observed small effect on the refolding reaction in the C11A/C27S variant is a consequence of large and compensating changes in the entropy and enthalpy of activation. Structural interpretation of the kinetic data suggests that formation of the beta-hairpin stabilized by the C11-C27 disulfide bond forms in the rate-limiting step of the refolding process. The interactions between the outer strands of the beta-sheet connected by the C45-C73 disulfide bond, in contrast, are made late in refolding. These results support the idea that beta-hairpins are initiation sites for beta-sheet formation and that additional strands are added late in the folding process.

Disulfides↗
PubMed1996

Native-like beta-structure in a trifluoroethanol-induced partially folded state of the all-beta-sheet protein tendamistat.

The effect of trifluoroethanol (TFE) on the structure of the all-beta-sheet protein tendamistat was investigated. At low concentrations TFE induces cooperative loss of the native tertiary structure leading to a partially folded state. The loss of specific side-chain interactions in the transition from the native state of the TFE-induced state is demonstrated by the disappearance of the CD bands in the aromatic region, a reduced chemical shift dispersion of the one-dimensional 1H NMR spectrum and a broad, uncooperative thermal unfolding transition of the partially folded state. An increased line-width of the NMR bands in the TFE state compared with the unfolded state suggests the presence of multiple, rapidly interconverting conformations. Hydrogen-exchange studies of amide proteins in the TFE state reveal the existence of defined hydrogen bonds at the same locations as in the native state, but with largely reduced stability. This suggests the presence of most of the native beta-sheet structure. These results are supported by Fourier transformed IR measurements, which show nearly the same amount of beta-structure in the TFE state and in the native state. Far UV CD spectroscopy suggests the induction of some alpha-helical structure upon addition of TFE, which appears to be located mainly in regions corresponding to loops or random structure in the native state and which seems to represent fluctuating conformations with preferred backbone angles rather than stable, hydrogen-bonded alpha-helices. These results show that stable non-local interactions, as they occur in beta-sheets, can form in the absence of specific side-chain interactions. The presence of a subset of the native long-range interactions and the absence of stable non-native interactions suggests that the observed partially folded state might represent an early intermediate on a hierarchical folding pathway of tendamistat.

Hydrogen Bonding↗
PubMed1995

Gene-targeted inhibition of transactivation of human immunodeficiency virus type-1 (HIV-1)-LTR by antisense oligonucleotides.

We have used an in vitro approach to study the efficiency of antisense oligonucleotides in inhibiting LTR-(HIV-1)-directed CAT expression catalyzed by tat protein, the functional protein of the transactivator gene. We selected the target sequence localized near the 5' end of the tat mRNA. The following conclusions can be drawn from the data presented here: a) Antisense oligonucleotides modified by conjugation of cholesterol at the 3' end have a severalfold higher inhibitory response, b) inhibitory response is dependent on the mode of introducing oligonucleotides, and c) the inhibition by antisense oligonucleotides is sequence specific and directed towards the targeted region. This approach could be useful for targeting functional regions of regulatory gene products and designing gene-targeted inhibitors of virus replication.

Animals↗
PubMed1990

Synthesis and secretion of hirudin by Streptomyces lividans.

To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the alpha-amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.

Amino Acid Sequence↗
PubMed1989

Primary structures of new 'iso-hirudins'.

Crude hirudin (12.7 U/micrograms), a complex mixture of polypeptides obtained from the leech, could be separated by microbore HPLC. A combination of amino acid analysis, N-terminal microsequencing and chemical as well as enzymatic fragmentation made the primary sequence of the new isohirudins Ia-IIIb' accessible. The biological activity determined in the thrombin inhibition test showed a comparable value for all of these compounds. The results presented address the question as to whether these isohirudins are true mutations from a family of genes or a family of leeches.

Amino Acid Sequence↗
PubMed1988

Gene synthesis.

Explore the source record for details and available documents.

Cloning, Molecular↗
PubMed1987

Inotropic and electrophysiological effects of 8-substituted cyclic AMP analogues on guinea-pig papillary muscle.

The inotropic potencies of 8-substituted cyclic AMP analogues, applied as sodium salts and in form of benzyl esters, were determined in isolated guinea-pig papillary muscles contracting isometrically at a frequency of 0.2 Hz. Half-maximally effective concentrations, EC50, for the positive inotropic effect of 8-substituted cyclic AMP (sodium salt) increased in the order 8-(4-chloro-phenyl)thio-cyclic AMP, 8-tertiary-butyl-thio-cyclic AMP, 8-benzyl-seleno-cyclic AMP, 8-benzyl-thio-cyclic AMP, 8-methyl-thio-cyclic AMP, 8-bromo-cyclic AMP. Neutralization of the phosphate hydroxyl residue of 8-substituted cyclic AMP by a benzyl group yielded cyclic AMP benzyl esters (cAMP-O-Bn) which were 30 to 100 times more potent than the respective cyclic AMP salts. Cyclic AMP derivatives with a 8-(4-chloro-phenyl)thio- or a 8-tertiary butyl-thio substituent showed comparatively high inotropic potencies. The intrinsic activity was uniformely the same for all 8-substituted cyclic AMP derivatives and equalled that of isoprenaline. As measured by octanol/water partitioning (log P), the increase in lipophilicity of 8-substituted cyclic AMP by esterification with a benzyl group was 7000-fold for 8-bromo-cyclic AMP, 5000-fold for 8-methyl-thio-cyclic AMP, and approximately 1000-fold for the other derivatives. Within the series of benzyl esters, differences in lipophilicity were small. The positive inotropic effect of 8-substituted cyclic AMP analogues was accompanied by a shortening of contraction duration, mainly due to an abbreviation of relaxation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1987

Influence of phosphodiesterase inhibition and of carbachol on inotropic effects of 8-substituted cyclic AMP analogues.

The influence of phosphodiesterase inhibitors and of carbachol on the positive inotropic effect of 8-substituted cyclic AMP analogues was studied on isometrically contracting guinea-pig papillary muscles driven at a rate of 0.2 Hz. In muscles from reserpine-pretreated animals, the phosphodiesterase inhibitors 3-isobutyl, 1-methyl xanthine (IBMX; 20 mumol/l) and papaverine (10 mumol/l) shifted the concentration-effect curves of 8-substituted cyclic AMP benzyl esters to the left, decreasing the EC50 by a factor of 10 to 25. In the presence of IBMX (5 and 20 mumol/l) or papaverine (10 mumol/l), the slopes of the concentration-effect curves of 8-substituted cyclic AMP benzyl esters became flatter. The positive inotropic effect and the increase in Vmax, overshoot and duration of slow action potentials induced by cyclic AMP analogues were not affected by carbachol (0.1-10 mumol/l). In the presence of IBMX (20 mumol/l), however, carbachol (3 mumol/l) antagonized the positive inotropic effect of 8-substituted cyclic AMP derivatives, shifting the EC50-values by a factor of 3 to the right. Cyclic AMP content determined by radioimmunoassay in individual papillary muscles was raised 1.22 and 1.63-fold in the presence of 3 and 20 mumol/l IBMX. Isoprenaline (0.1 mumol/l) induced an increase in cyclic AMP content which was not significantly different from that produced by 20 mumol/l IBMX, but in contrast to the phosphodiesterase inhibitor enhanced force of contraction by 17.7 mN as compared to 1.5 mN obtained with 20 mumol/l IBMX. The findings are consistent with a model that describes the interaction between IBMX and cyclic AMP analogues as an additive effect with only endogenously accumulated cyclic AMP (due to phosphodiesterase inhibition) being involved in the negative inotropic effect of carbachol. From the failure of carbachol to affect the positive inotropic effect of cyclic AMP analogues, it is concluded, that cyclic AMP derivatives do not act as phosphodiesterase inhibitors, and that the well-known negative inotropic effect of carbachol in the presence of cyclic AMP-elevating drugs does not occur at a step beyond cyclic AMP accumulation.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.

Amino Acid Sequence↗
PubMed1984

[Parenteral administration of amino acids. Reference values--study technic--interpretation of the study results--amino acid pattern as a criterion of the regulation of homeostasis].

The concentrations of free amino acids were measured in lithium-heparinat-plasma with a standardized quality controlled analytical procedure in 200 healthy persons (blood donors). The distribution of the measured values were statistically evaluated in the total group and differentiated in the male and female subcollective. The absolute total concentrations of the free amino acids vary in a broad range between 1.7 and 4.4 mmol/l. This large range is also seen for the single amino acid. In a new system for documentation and interpretation of analytical data the measured values are collected and mean, median, standard deviation, mini-max-range and 95% confidence interval are automatically computed. On this basis of data a physiological reference range is defined. The broad variability of the absolute concentrations is not found in the relative composition of the pattern. The relations of the amino acid concentrations which are important for the quality of the cellular support are very constant. Their importance is caused by the fact that only few different transport systems exist for at least 20 amino acids, which means that the different amino acids are competing at the same system for cellular uptake. The essential of the results in this study is that the liver guarantees the constancy of the physiological pattern also in cases where the exogenous supply differs from the characteristics of elimination of the single amino acid from blood. Only when the oxidative capacity of the liver is insufficient as in pathological states like infections pathological concentrations are found. In these cases the true change is undiscovered when the absolute concentrations are considered, because the insufficient functions of the liver causes initially high total concentrations. For the interpretation of an analysis of amino acids in blood plasma two main criteria are necessary: the total concentration of amino acid characterizes the quantitative support while the concentration pattern determines the quality of the cellular uptake.

Adult↗
PubMed1983

3'-O-methylated analogs of 2-5A as inhibitors of virus replication.

We have explored analogs of 2'-5'-linked andeylic acid trimer (2-5A): 3'-O-methylated 2-5A, 2'-end modified adenylate trimer with deoxyadenosine or araadenine, methyl phosphonate and methyl phosphorotriester analogs as potential antiviral agents. For the treatment of virus infections, 2-5A and its analogs may serve in lieu of interferon, however, the use of 2-5A has two serious limitations: it is presumed to be impermeable to most cells, and moreover, cellular enzymes rapidly degrade it. Methylated analogs of 2-5A core strongly inhibited virus growth when added directly to cells in culture. 2'-End modified adenylate trimer with araadenine also inhibited virus growth, however, neither 2-5A nor other analogs showed any significant antiviral activity. The inhibition of virus growth was not due to the toxic effect of these compounds on cell growth as they had no inhibitory effect on the growth of uninfected cells.

Adenosine Monophosphate↗
PubMed1983

Time course of the increase in the myocardial slow inward current after a photochemically generated concentration jump of intracellular cAMP.

Voltage-clamped atrial trabeculae from bullfrog hearts were exposed to membrane-permeant photolyzable o-nitrobenzyl esters of cAMP and cGMP. UV flashes produced intracellular concentration jumps of cAMP or cGMP. With the cAMP derivative, flashes resulted in an increased slow inward current (Isi), producing a broadened action potential. The Isi reached a maximum 10-30 sec after the flash and decreased over the next 60-300 sec. The first increases were observable within 150 msec; this value is an upper limit imposed by the instrumentation. Responses to flashes lasted longer at higher drug concentrations and in the presence of the phosphodiesterase inhibitor papaverine; effects of flashes developed and decreased faster at higher temperature. Although the amplitude of the Isi was increased, its waveform and voltage sensitivity were not affected. Intracellular concentration jumps of cAMP failed to affect the muscarinic K+ conductance. There were no observable effects of cGMP concentration jumps. The data confirm (i) that cAMP regulates the Isi and (ii) that the 5- to 10-sec delay between application of beta-agonists and the onset of positive inotropic effects, observed in previous studies, has been correctly ascribed to events prior to the interaction between cAMP and protein kinase.

Animals↗
PubMed1977

Synthesis, structure, and reactivity of adenosine cyclic 3',5'-phosphate benzyl triesters.

A series of triesters of adenosine cyclic 3',5'-phosphate was synthesized by treatment of the free acid with various diazoalkanes (R=H, CH3, C6H5,0-NO2C6H4, p-NO2C6H4, p-CH3C6H4). The resulting diastereomeric mixtures were separated into their axial and equatorial components. Hydrolysis of the compounds was examined as well as photolysis of the photolabile o-nitrobenzyl ester. All compounds were then tested for their ability to activate the cAMP-dependent protein kinase and for their ability to serve as a substrate for the cAMP phosphodiesterase showing almost no effect on either enzyme. In a biological assay the benzyl triesters were able to penetrate into C 6 rat glioma cells and to induce the typical morphological alteration of the cell shape known for high cellular levels of cAMP. It was concluded that the benzyl triesters of cAMP are useful derivatives which can be efficiently and specifically converted to the parent nucleotide. Benzyl derivatives of biologically active phosphodiesters may provide a useful tool for study in biology and pharmacology.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

[Rapidly debilitating disease with generalized lymphadenopathy, skin involvement and interstitial pulmonary infiltration (report of three cases)].

Generalized but well-circumscribed lymphadenopathy and rash-like skin changes were observed in three men, aged 58 to 75 years. There was a reticular appearance in the chest X-ray. Dyspnoea, weakness, marked weight loss, changing but marked lymphopenia, markedly increased blood-sedimentation rate, and an always negative Tine test were present in all three. Despite antibiotics, cytostatic drugs and prednisolone the disease quickly ended fatally with high fever, general debilitation and pneumonia. Post-mortem examination revealed diffuse lymphatic hyperplasia with plasma-cell infiltration in the lymph nodes, tonsils and lymphatic tissue of the intestines, and diffuse hyperplasia of the endothelial venules, together with basophilic blast cells, eosimophilic granulocytes and reticulum cells with broad nucleoli. The spleen was normal or enlarged. Spleen, lung tissue and lymphatics, the skin in the area of the small vessels, hair follicles and sweat glands contained lymphocytes, plasma cells and eosinophilic leucocytes. The spleen, if enlarged, also had focal necroses in its periarteriolar septa. In two cases electrophoresis revealed beta-globulin poorly demarkated from gamma-globulin, doubling and increase. In the third case, IgA was markedly increased with one each monoclonal IgG1 und IgA, and corresponding shift in the chi/lambda relationship in serum. Immunohistologically, lymphocytes were made up of about 20 percent IgG-containing and about 25 percent IgA-containing cells. Lymphopenia, if present, was associated with markedly reduced blast transformation of peripheral lymphocytes to phytohaemagglutinin. The granulocytes were defective (intracellular killing of Candida albicans and Staphylococcus aureus), while the nitrobluetetrazolium test was normal. There was RBC phagocytosis in the macrophages of bone marrow and in Kupffer cells of all three cases.

Aged↗