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.

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.

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.

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 "fallen lung with absent hilum" signs of complete bronchial transection.

A 19-year-old pedestrian who was the victim of a motor vehicle accident had a left pneumothorax with a large air leak. Despite a well-placed chest tube, a chest radiograph showed that the left lung had fallen down and away from the mediastinum and that no hilar structures were visible. This "fallen lung with an absent hilum" is considered virtually diagnostic of complete mainstem bronchus transection.

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.

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.

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.

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.

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells.

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.

Analysis of a soluble mutant des-methionine interleukin-2 receptor alpha chain (Tac protein) produced by transfected mammalian cells.

By using recombinant DNA technology the cytoplasmic and trans-membrane domain of the human interleukin-2 receptor alpha chain (IL-2R alpha, Tac) and of a mutant protein lacking methionine-residues 18, 25, 44, 88, 92, 126, 149, 167, 205, and 209 (des-Met IL-2R alpha) encoded by a chemically and enzymatically synthesized gene, were deleted. This leads to secretory expression of soluble wild-type and des-Met mutant Tac protein of 42-45 kDa after transfection of BHK-21 cells. Transfectants secreted up to 1.

Secretory synthesis of human interleukin-2 by Streptomyces lividans.

To study the ability of Streptomyces lividans to produce heterologous proteins by secretion, we directly fused DNA encoding the leader peptide of the alpha-amylase inhibitor, tendamistat, produced by Streptomyces tendae, with DNA encoding the mature part of interleukin-2 (IL-2). Such cloned fusion constructs are translated in S. lividans, in spite of the quite different codon usage.

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.

Synthesis and selective cleavage of oligodeoxyribonucleotides containing non-chiral internucleotide phosphoramidate linkages.

Oligodeoxynucleotides containing phosphoramidate internucleotide links 3'-OP(O)NH-5' have been prepared using standard solid phase phosphoramidite techniques. For the incorporation of the phosphoramidate linkages we have used monomer as well as dimer building blocks. With the monomer 3'-phosphoramidite building blocks, which are derived from 5'-amino-2',5'-dideoxynucleosides, it is possible to incorporate phosphoramidate links into specific positions within an oligodeoxynucleotide.

Biliary carcinoma: CT evaluation of extrahepatic spread.

The authors studied the computed tomographic (CT) images obtained in 56 patients with pathologically proved biliary cancer and 75 patients with no evidence of biliary disease, attempting to define the normal anatomy of the lymphatic system draining the bile ducts and the prevalence of extrahepatic spread of primary biliary cancer into these retroperitoneal planes. Of 20 patients with gallbladder cancer, 14 (70%) had proved adenopathy and nine (45%) had peritoneal spread at presentation, and another three later developed carcinomatosis. Of 22 patients with proximal cholangiocarcinoma, 16 (73%) had nodal involvement at presentation, four later developed adenopathy, and five had peritoneal dissemination.

Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity.

A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate.

Synthesis and structure assignments of amide protected nucleosides and their use as phosphoramidites in deoxyoligonucleotide synthesis.

The syntheses of several amide protected deoxyguanosine- as well as thymidine nucleosides are described. These compounds were synthesized according to the Mitsunobu reaction and Michael addition. In contradiction to previous studies we have discovered that the Michael addition gives only products derived from N-alkylation.

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45.

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.

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.

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.

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.

[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.