Biomedical subjects

J W Engels

Publications and source records attributed to J W Engels.

At least 37 records · Page 2Linked to original sources

PubMed1997

Synthesis and binding properties of oligodeoxynucleotides containing phenylphosphon(othio)ate linkages.

A method for the synthesis of chimeric oligodeoxynucleotides comprised of phosphodiester and phenylphosphonate [3'-O-P(= O)(C6H5)-O-5'] or phenylphosphono-thioate [3'-O-P(= S)(C6H5)-O-5'] linkages has been developed. Synthesis was performed using suitably protected nucleoside phenylphosphonamidites as building blocks following an adjusted solid-phase phosphoramidite synthesis protocol. The new oligodeoxy-nucleotide analogues were characterized by electrospray ionization- and matrix-assisted laser desorption mass spectrometry, as well as by 31P NMR spectroscopy. Additionally, their binding properties to complementary oligodeoxynucleotides has been studied.

Chromatography, High Pressure Liquid↗

PubMed1996

Influence of specific signal peptide mutations on the expression and secretion of the alpha-amylase inhibitor tendamistat in Streptomyces lividans.

The Streptomyces alpha-amylase inhibitor tendamistat is secreted by a signal peptide with an amino-terminal charge of +3. To elucidate the influence of the charged residues on protein secretion in Streptomyces, the amino-terminal charge was varied from +6 to neutral net charge. The effects of charge variation were analyzed in combination with three Streptomyces promoters and two transcriptional terminators. Introduction of additional positive charges significantly decreased the amount of secreted tendamistat. On the contrary, a charge reduction to +2 resulted in the doubling of inhibitor production. After exclusion of transcriptional effects, the observed alterations of inhibitor secretion by the mutants with a charge of +6 to +2 were attributed to a modulation of precursor synthesis. Furthermore, a tight coupling of synthesis and export was stated. Charge reduction to +1 or neutral charge generally reduced the yield of secreted tendamistat, yet remarkable differences were found for mutants with identical net charge. Elimination of the positive charge at a defined position resulted in the release of tendamistat precursor protein, which suggested a specific uncoupling of synthesis and translocation.

Amino Acid Sequence↗

PubMed1996

Synthesis and properties of new antisense oligodeoxynucleotides containing benzylphosphonate linkages.

Synthetic antisense oligonucleotides are widely used as inhibitors of gene expression in cultured cells and have been proposed as potential therapeutic agents. In recent years, a great deal of effort has been directed toward the synthesis of analogs with an altered phosphodiester linkage to improve the stability of duplex formation, to improve the cellular uptake, and to decrease the rate of degradation of oligonucleotides by nucleases that cleave the phosphodiester linkage. We report here on the synthesis of oligonucleotides containing benzylphosphonate linkages. For this purpose, we prepared four fully protected benzylamidites and incorporated them into oligonucleotides using an automated DNA synthesizer. Compared with unmodified oligonucleotides and methylphosphonates, the benzylphosphonates are more stable against nucleases and exhibit a higher lipophilicity. Incorporation of benzylphosphonate linkages only slightly weakens the thermal stability of the duplex. The 18-mer oligonucleotide 5' A*T*GTT*GCCC*CATC*ATA*AA 3' with seven benzylphosphonate linkages (*) directed against the start of the pre-S-region of duck hepatitis B virus resulted in a 40% inhibition of viral replication at a concentration of 1.5 mumol.

Animals↗

PubMed1995

Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.

The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗

PubMed1995

6-Oxocytidine a novel protonated C-base analogue for stable triple helix formation.

2'-O-Methyl-3'-O-phosphoramidite building blocks of 6-oxocytidine 6 and its 5-methyl derivative 7, respectively, were synthesized and incorporated via phosphoramidite chemistry in 15 mer oligodeoxynucleotides [d(T72T7), S2; d(T73T7), S3] to obtain potential Py.Pu.Py triplex forming homopyrimidine strands. UV thermal denaturation studies and CD spectroscopy of 1:1 mixtures of these oligomers and a 21 mer target duplex [d(C3A7GA7C3)-d(G3T7CT7G3), D1] with a complementary purine tract showed a nearly pH-independent (6.0-8.0) triple helix formation with melting temperatures of 21-19 degrees C and 18.5-17.5 degrees C, respectively (buffer system: 50 mM sodium cacodylate, 100 mM NaCl, 20 mM MgCl2). In contrast, with the corresponding 15mer deoxy-C-containing oligonucleotide [d(T(7)1T7), S1] triplex formation was observed only below pH 6.6. Specificity for the recognition of Watson-Crick GC-base pairs was observed by pairing the modified C-bases of the 15mers with all other possible Watson-Crick-base compositions in the target duplex [d(C3A7XA7C3)-d(G3T7YT7G3), X = A,C,T; Y = T,G,A, D2-4]. Additionally, the Watson-Crick-pairing of the modified oligomers S2 and S3 was studied.

Base Sequence↗

PubMed1995

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients.

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted from delta TCS-1-positive T lymphocytes. At concentrations of 1.25-10 microM, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, of delta TCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.

Antigens, CD↗

PubMed1994

Isolation and refolding of a mutant methionine-free interleukin-2-receptor alpha chain synthesized as a fusion protein in Escherichia coli.

A soluble domain of the interleukin(IL)-2 receptor, the alpha chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine-free IL-2 receptor alpha chain (synIL-2R alpha) after BrCN cleavage and renaturation of the crude cleavage material, although the alpha chain is expressed as a deglycosylated, methionine-free protein. The soluble receptor comprises amino acids 1-219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL-2 and monoclonal antibodies as ligands. Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions. The deletion of a non-bridging cysteine at position 192 in the synIL-2R alpha did not affect the renaturation yield of the receptor protein. Additionally a cysteine-free and methionine-free beta-galactosidase derivative was fused to the soluble synIL-2R alpha derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN-cleavage material. It is suggested that cysteine impurities from cyanogen-bromide-cleaved peptides might interfere seriously with the refolding process of the synthetic IL-2 receptor alpha-subunit.

Amino Acid Sequence↗

PubMed1994

Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.

Base Composition↗

PubMed1994

The nuclear-magnetic-resonance solution structure of the mutant alpha-amylase inhibitor [R19L] Tendamistat and comparison with wild-type Tendamistat.

The recombinant mutant alpha-amylase inhibitor [R19L]Tendamistat, with Arg19 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence-specific 1H-NMR assignments, 845 nuclear Overhauser effect upper-distance constraints and 156 dihedral angle constraints were collected using two-dimensional homonuclear 1H-NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild-type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & Wüthrich, K. (1988) J. Mol. Biol. 204, 675-724]. For each protein a group of 20 conformers represents a well-defined solution structure, with average root-mean-square-distance values for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strictly localized near the single amino acid substitution in the presumed active site -Trp18-Arg(Leu)-Tyr-, i.e. Leu19 and Tyr20 are more precisely defined in the solution structure of [R19L]Tendamistat than the corresponding residues Arg19 and Tyr20 in wild-type Tendamistat.

Amino Acid Sequence↗

PubMed1993

High yield fermentation and purification of Tendamistat disulphide analogues secreted by Streptomyces lividans.

In our studies of structure-function correlation of polypeptides we used Tendamistat (TM), an alpha-amylase-inhibitor of Streptomyces tendae, as a model to investigate the influence of different mutants on the expression and secretion of the protein. In addition, we examined the influence of replacing the two disulphide-bridges that stabilize the two-loop structure of the whole protein. The single mutants C27S, C27T, C45A, the double mutants C11A/C27A, C11A/C27S, C11A/C27T, C11A/C27L, C45/C73A and the fourfold mutant C11A/C27A/C45A/C73A were prepared. The mutated TM gene was expressed in S. lividans TK 24, which secretes the active form of the inhibitor into the culture medium. Compared with the wild-type, the double-mutated TM derivatives show an increase in secreted protein by a factor of two to ten. In contrast, the single-mutated inhibitor analogues show the reverse effect. In order to examine the influence of temperature and culture media on the production of protein derivative we used the most unstable C11A analogue. Our expression studies at 10, 19, 28 and 37 degrees C established 19 degrees C as the optimal temperature for production of the protein derivatives. The correlation between the stability and secretion of TM is discussed in the context of our knowledge of protein translocation in bacteria. Based on these experiments we optimized the fermentation parameters, isolated TM analogous on a large scale, and verified them.

Amino Acid Sequence↗

PubMed1992

Cell type-specific tyrosine phosphorylation of IL-2 receptor beta chain in response to IL-2.

Functional activities of the IL-2 receptor (IL-2R) beta chain exogenously expressed on lymphoid and non-lymphoid cells were examined in terms of phosphorylation of IL-2R beta and cell growth. Lymphoid MOLT-4 and its transfectants expressing IL-2R beta either alone or with IL-2R alpha chain were found to be rapidly phosphorylated predominantly at tyrosine residues of IL-2R beta and to be affected in their growth in an IL-2-dependent manner. In contrast, IL-2 induced neither phosphorylation of IL-2R beta nor cell growth in non-lymphoid transfectants derived from COS7, HeLa and L929, even though they acquired the IL-2 binding ability when coexpressed as IL-2R beta and IL-2R alpha. These results suggest that IL-2 induces activation of a tyrosine kinase possibly associated with IL-2R beta in a cell type-specific manner.

Cell Division↗

PubMed1992

Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity.

Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.

Amino Acid Sequence↗

PubMed1992

Promoter constructions for efficient secretion expression in Streptomyces lividans.

Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S. tendae, in order to study secretion-expression in S. lividans using a pIJ702 plasmid vector system. Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency. The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S. fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus. In addition, we tested the thiostrepton-inducible tipA promoter from S. lividans in our Tendamistat secretion system. The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation. The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter. The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion. Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.

Biotechnology↗

PubMed1992

Antisense oligodeoxynucleotide: inhibitor of splicing of mRNA of human immunodeficiency virus.

Antisense oligodeoxynucleotide (ODN), which are directed against the splice acceptor site of exon II of the regulatory gene tat of the human immunodeficiency virus type 1 (HIV-1), have been described. These 20-mer ODN's displayed moderate anti-HIV activity in vitro. Using the same antisense ODN (termed ODN-2), which was additionally modified and protected both at the 3'- and the 5'-terminus by two phosphorothioate internucleotide linkages, a strong anti-HIV activity (EC50: 2.7 micrograms/ml) could be measured in the HIV-1/CEM- and HIV-1/HeLa-T4+ cell system. The analogous ODNs which were protected only at one end were either inactive (up to 10 micrograms/ml) or displayed a low antiviral activity. Time kinetic studies revealed that the antisense ODN-2 reduced the release of HIV-1 already after an incubation time of 1 h. By applying S1 nuclease protection procedures, it could be established that the antisense ODN-2 inhibited splicing of high molecular weight transcript to the 2-kb tat mRNA in HIV-1-infected CEM cells. Transfection experiments with pU3R-III chloramphenicol acetyltransferase expression vector in HeLa-T4+ cells revealed that the antisense ODN-2 blocked the Tat protein-mediated transactivation process. In co-transfection experiments using pSV2tat72 or scrape loading studies with purified Tat, the transactivation was restored. These data indicate that the selected antisense ODN-2 displays its anti-HIV effect by blocking the splicing process leading to the functional 2-kb tat mRNA.

Antiviral Agents↗

PubMed1991

Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5'-phosphorothioate linkage.

A self complementary oligodeoxynucleotide dodecamer containing an achiral bridged 5'-phosphorothioate linkage 3'-O-P-S-5' has been prepared using the solid phase phosphoramidite procedure. For the incorporation of the phosphorothioate link we have used a 5'-(S-trityl)-mercapto-5'-deoxythymidine-3'-phosphoramidite. After coupling this building block the S-trityl group was removed by silver ions. The free thiol moiety was then coupled with a standard phosphoramidite in the presence of tetrazole. After oxidation of the 5'-phosphorothioite with iodine/water the synthesis was continued with standard building blocks up to the desired dodecamer. This backbone modified dodecamer can be cleaved selectively and quantitatively at the P-S bond by silver or mercuric ions under very mild conditions.

Base Sequence↗

PubMed1991

Backbone modified oligodeoxynucleotides: stereoselective synthesis of methylphosphonates and HIV inhibitory capacity of phosphorothioates.

Oligonucleotides bearing phosphorothioate linkages of the HIV tatIII splice acceptor site were synthesized by automated solid phase synthesis. Especially 5'- and 3'-end capped thioates of the sequence 5'-ACACCCAATTCTGAAAATGG-3' show microM inhibition of HIV replication. ODN-methylphosphonates of defined stereochemistry were obtained with suitably modified proline phosphonamidite derivatives as monomeric building blocks. Asymmetric induction for nucleosidephosphonamidates up to 5:1 (Rp:Sp rsp. Sp:Rp depending on configuration of proline-moiety) could be reached. The intermediate phosphonamidite can be further reacted to dinucleoside methylphosphonates of enriched diastereomeric excess.

Antiviral Agents↗

PubMed1990

Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein.

Several fusion proteins of our previously chemically synthesized gene encoding the interleukin-2-receptor alpha subunit (IL-2R alpha or Tac protein) were constructed. They were designed in order to be cleavable by cyanogen bromide. Thus, the original internal methionines of the IL-2R alpha were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions. Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid-labile Asp-Pro bond. Direct C-terminal fusion of total beta-galactosidase and portions thereof did not result in substantial amounts of the expected construct. Ternary fusions consisting of beta-galactosidase domains N- and C-terminally fused to the mutant synthetic methionine-free interleukin-2 receptor alpha subunit (synIL-2R alpha) yielded inclusion bodies amounting to 4-7% of the total protein. This first overexpression of a type I membrane receptor can be rationalized by the known beta-galactosidase structure models. The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL-2R alpha detected by Western blot analysis.

Amino Acid Sequence↗

PubMed1990

Diastereomeric dinucleoside-methylphosphonates: determination of configuration with the 2-D NMR ROESY technique.

The determination of configuration at phosphorus in diastereomeric dinucleoside-methylphosphonates having the -O-P(= O)(-CH3)-O- internucleotide linkage with the NOE derived ROESY NMR technique is described for ApT, TpT, ApA, TpA and CpG. For this purpose ROE's from the P-CH3 group to the protons in the nearest neighbourhood were measured. These ROE's are different within diastereomeric pairs of a dimer enabling us to deduce the individual configuration. The validity of the method is proven in comparison with dimers of known configuration (ApT, TpT). Together with a recently published diastereoselective synthesis method a more homogeneous picture between physical properties and the corresponding configuration is provided. There is an improvement in our knowledge about the stereochemistry of these substances which could not be deduced from the data known before.

Dinucleoside Phosphates↗