Revised interpretation of the immunological results obtained with pneumococcal polysaccharide 17F derived synthetic di-, tri- and tetrasaccharide conjugates in mice and rabbits.

Recently, the structure for pneumococcal polysaccharide (PS) 17F has been revised. Based on the former PS structure, immunogenicities of PS 17F derived synthetic di-, tri- and tetrasaccharide conjugates have been reported in mice. Here, we present additional data on the immunogenicities of these conjugates in rabbits and re-evaluate the immunogenicity results in the light of the revised PS 17F structure.

Solution-phase and solid-phase synthesis of novel transition state inhibitors of coagulation enzymes incorporating a piperidinyl moiety.

2-Amino-3-piperidin-4-yl-propionic acid containing peptidomimetics are potent protease inhibitors when combined with an appropriate keto-thiazole or keto-carboxylic acid moiety. A novel P1 residue in factor Xa and thrombin inhibitors has been found resulting in IC50 values as low as 0.048 microM, a factor of ten more potent than Argatroban. Starting with non-chiral synthetic routes, a new stereospecific route was developed as well as a new solid-phase method.

Tyrosine kinase activity of purified recombinant cytoplasmic domain of platelet-derived growth factor beta-receptor (beta-PDGFR) and discovery of a novel inhibitor of receptor tyrosine kinases.

Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.

Alpha-sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of laminin.

Schwann cells play an important role in peripheral nerve regeneration. Here, we report the effect of alpha-sialyl cholesterol (alpha-SC), a derivative of the sialic acid-containing natural gangliosides, and the cytostatic agents, cisplatin, taxol and vincristine on the laminin production in Schwann cell cultures isolated from rat sciatic nerves. Laminin, one of several extracellular matrix components produced by Schwann cells, is known to potentiate axonal outgrowth. Laminin content was increased by alpha-SC, starting at 7.0 micrograms/ml with a maximal effect at 22.4 micrograms/ml (30%, P < 0.001). The three cytostatic drugs, dose-dependently reduced laminin content in Schwann cell cultures: (1) cisplatin at a threshold dose of 2 micrograms/ml (-26.4%, P < 0.001); (2) taxol, starting at a dose of 1 ng/ml (-8.0%, P < 0.05); and (3) vincristine, starting at 0.5 ng/ml (-5.9%, P < 0.05). Cultured Schwann cells were incubated with cytostatic drugs in combination with increasing amounts of alpha-SC and it was found that, depending on the cytostatic drug concentration used, alpha-SC could reduce or completely prevent the cytostatic drug-induced reduction of laminin in Schwann cell cultures. Co-treatment with alpha-SC also reduced part of the morphological changes caused by the cytostatic drugs. alpha-SC did not counteract the anti-proliferative effect of the cytostatic drugs on K-562 human erythroleukemia cells. In conclusion, alpha-SC increased laminin content in Schwann cell cultures and protected Schwann cell cultures against the decrease of laminin by cytostatic drugs without interfering with the anti-proliferative potential, suggesting that alpha-SC may have clinical use in protecting cancer patients against the neurotoxic effects of cytostatic drugs.

Protein-conjugated synthetic di- and trisaccharides of pneumococcal type 17F exhibit a different immunogenicity and antigenicity than tetrasaccharide.

Overlapping synthetic disaccharide, trisaccharide and tetrasaccharide, derived from pneumococcal polysaccharide type 17F (PS17F), were coupled to keyhole limpet haemocyanin (KLH). The conjugates were tested in mice. The disaccharide-KLH and especially trisaccharide-KLH, in combination with Quil A, induced high titres of high-avidity anti-PS17F IgG. Both conjugates protected mice against challenge with Streptococcus pneumoniae 17F. Tetrasaccharide-KLH, although able to elicit anti-tetrasaccharide antibodies, induced a minimal non-protective anti-PS17F IgG response of low avidity. The tetrasaccharide-KLH conjugate, in contrast to the other conjugates, failed to bind rabbit anti-PS17F IgG.

Iodonium ion-assisted synthesis of a haptenic tetrasaccharide fragment corresponding to the inner cell-wall glycopeptidolipid of Mycobacterium avium serotype 4.

Condensation of ethyl 2,4-di-O-benzoyl-1-thio-alpha-L-rhamnopyranoside with ethyl 3-O-benzyl-4-O-chloroacetyl-2-O-methyl-1-thio-beta-L-fucopyranoside in the presence of iodonium di-sym-collidine perchlorate afforded exclusively ethyl 2,4-di-O-benzoyl-3-O-(3-O-benzyl-4-O- chloroacetyl-2-O-methyl-alpha-L-fucopyranosyl)-1-thio-alpha-L-rhamnop yra noside. This disaccharide derivative was extended at C-1 with 3-benzyloxycarbonylaminopropyl 6-deoxy-3,4-O-isopropylidene-alpha-L- talopyranoside, using N-iodosuccinimide and triflic acid as the catalyst, to furnish 3-benzyloxycarbonylaminopropyl 6-deoxy-2-O-[2,4-di-O-benzoyl-3-O-(3-O-benzyl-4-O-chloroacetyl-2-O-me thy l- alpha-L-fucopyranosyl)-alpha-L-rhamnopyranosyl]-3,4-O-isopropylidene-alp ha-L- talopyranoside (20). Selective removal of the chloroacetyl group from 20, followed by condensation with ethyl 2,3-di-O-benzoyl-4-O-methyl-1-thio-alpha-L- rhamnopyranoside in the presence of the same thiophilic promoter, yielded a fully protected tetrasaccharide derivative. Deprotection of the latter gave the target compound 3-aminopropyl 6-deoxy-2-O-[3-O-[2-O- methyl-(4-O-methyl-alpha-L-rhamnopyranosyl)-alpha-L-fucopyranosyl]-alpha -L- rhamnopyranosyl]-alpha-L-talopyranoside (1).

Specific recognition of antibody-oligonucleotide conjugates by radiolabeled antisense nucleotides: a novel approach for two-step radioimmunotherapy of cancer.

One of the major challenges in radioimmunotherapy is the specific delivery of radioisotopes to tumor cells while minimizing normal tissue radiation. In this respect, the application of two-step pretargeting schemes generally leads to more favorable tumor to normal tissue uptake ratios than direct administration of radioimmunoconjugates. In this study, we present the specific hybridization of complementary DNA fragments as a novel recognition mechanism in pretargeting. Briefly, our strategy involves first administration of antibody-DNA conjugate, followed by targeting with radiolabeled complementary DNA (antisense DNA). Complementary oligodeoxynucleotides (14-mers, Tm = 57 degrees C), in which part of the phosphodiesters has been replaced by methylphosphonates (to ensure stability against nucleases), were prepared on a DNA synthesizer. The oligonucleotides were further derivatized via a uridine moiety at their 5'-end in such a way that radiolabeling or conjugation with antibodies could be accomplished. Both a murine IgG (anti-hCG) and the human anti-tumor IgM 16.88 were conjugated with one to three oligonucleotides via the heterobifunctional cross-linker SMCC. Incubation of these immunoconjugates with the radiolabeled antisense DNA revealed specific hybridization with the antibody-linked oligonucleotides. Antigen binding studies performed with antigen-coated matrices showed that the immunoreactivity of the antibody-DNA conjugate is preserved. Moreover, it is demonstrated that the radiolabeled DNA is still capable of hybridizing selectively with the oligonucleotides of the immunoconjugate, when the latter is bound to its antigen.

Detection of Aspergillus and Penicillium extracellular polysaccharides (EPS) by ELISA: using antibodies raised against acid hydrolysed EPS.

Species of the fungal genera Aspergillus and Penicillium produce immunologically active extracellular polysaccharides (EPS) in which galactofuranose residues are immunodominant. The antigenic determinant of the EPS of A. fumigatus, A. niger and P. digitatum could be removed by acid hydrolysis. Due to the hydrolysis of the EPS the immunological reaction between IgG anti-native EPS and hydrolysed EPS disappeared. Antibodies raised in rabbits against the acid hydrolysed EPS revealed new antigenic determinants that were exposed as a result of the acid hydrolysis. Immunological inhibitory experiments showed that the antibodies were no longer directed to galactofuranose residues. Enzyme Linked Immunosorbent Assay, carried out with antibodies raised against the acid hydrolysed EPS showed that the antibodies against the acid hydrolysed EPS were more species specific in comparison with the antibodies against the native EPS.

Synthesis of a substrate of protein kinase C and its corresponding phosphopeptide.

The syntheses of a protein kinase C (PKC) peptide substrate, H-Lys-Arg-Thr-Leu-Arg-OH, and a phosphopeptide analog of the synthetic substrate, H-Lys-Arg-Thr(P)-Leu-Arg-OH, are reported. PKC phosphorylates the peptide with an apparent KM of 0.30 +/- 0.04 mM and an apparent Vmax equal to one-tenth that of histone III-S. The synthesis of the phosphopeptide features a recently developed convenient phosphorylation procedure for serine and threonine using N,N-diethylamino-dibenzylphosphoramidite. A complete characterization of the PKC substrate and its corresponding phosphopeptide by C-H COSY 2D n.m.r. is included.

A Rapid and Reliable Method for the Detection of Molds in Foods: Using the Latex Agglutination Assay.

An agglutination test by using latex beads (0.8 µm diameter) coated with IgG from the antibodies against extra-cellular polysaccharide of Penicillium digitatum has been developed. As low as 5 to 10 ng/ml of the purified extra-cellular polysaccharide of the same species can be detected by this preparation. Analysis of culture filtrates from 25 different molds showed that the positive reactivity was obtained only with the species of genera Penicillium and Aspergillus . The application of this test was confirmed in testing the samples of spices and nuts. Further, the reliability could be enhanced by including specific blocker in the assay, the synthetic epitopes consisting of four ß (1-5)-linked D-galactofuranosyl residues.

(1----5)-linked beta-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species.

Aspergillus and Penicillium species produce extracellular polysaccharides which are immunologically active. Methyl beta-D-galactofuranoside interferes with the reaction between the polysaccharide antigens and the antibodies raised in rabbits. Of the different interlinked dimers of beta-D-galactofuranosides (1----2; 1----3; 1----5; 1----6) the (1----5) interlinked beta-D-galactofuranoside gave the highest inhibition. An increasing inhibitory effect of di-, tri-, tetra-, penta-, hexa-, and heptamer of (1----5) interlinked beta-D-galactofuranosides was observed. It was noticed that the penta-, hexa- and heptamer of (1----5) interlinked beta-D-galactofuranosides were able to link antibodies raised against the extracellular polysaccharides produced by Penicillium species. The tetramer molecule was able to neutralize the binding of antibodies, which are naturally present in human sera, to the polysaccharides produced by Penicillium and Aspergillus species.

Fibrinogen lysine residue A alpha 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalyzed by tissue-type plasminogen activator.

In previous studies, we have shown that the stretch 148-197 of the fibrinogen A alpha chain plays a crucial role in the acceleration of the tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation. In this study we have synthesized parts of A alpha 148-197 and analogues thereof. We found that the peptides with sequences identical with A alpha 148-161 and A alpha 149-161 of human fibrinogen accelerate the plasminogen activation by t-PA, whereas the corresponding peptides in which lysine residues A alpha 157 had been replaced by valine or arginine had no accelerating capacity. Furthermore, succinylation of the lysine residue(s) in the synthesized peptides A alpha 148-161 and A alpha 149-161 leads to loss of accelerating action. These findings show that lysine residue A alpha 157 is crucial for the accelerating action of fibrin on the t-PA-catalyzed plasminogen activation.

gamma-Endorphin and schizophrenia: amino acid composition of gamma-endorphin and nucleotide sequence of gamma-endorphin cDNA from pituitary glands of schizophrenic patients.

The possibility has been mentioned that a change in the structure is responsible for the deviant behavioral activity of gamma-endorphin in extracts of postmortem brain and pituitary gland samples of schizophrenic patients. This paper describes the investigation of this possibility by means of: amino acid composition analysis of alpha- and gamma-endorphin isolated from a pituitary gland of a schizophrenic patient; and nucleotide sequence analysis of the gamma-endorphin coding region of pro-opiomelanocortin (POMC) mRNA from two other pituitary glands, using the primer extension method. Both methods require no more than a single pituitary to obtain reliable results. alpha- and gamma-endorphin were isolated from an acid extract by gel filtration and two subsequent HPLC steps. In addition, the gamma-endorphin region of beta-endorphin was analyzed by enzymatic cleavage of beta-endorphin and isolation of the resulting fragment. Single-stranded gamma-endorphin cDNA was synthesized by reverse transcriptase using total cellular pituitary RNA and a 5' 32P-labeled oligodeoxyribonucleotide primer (20-mer) hybridizing close to the gamma-endorphin coding region of POMC mRNA. Single-stranded cDNA was digested with restriction enzyme HaeIII which generated a 148 nucleotides long radioactive cDNA fragment containing the gamma-endorphin cDNA sequence. The sequence of the 148 nucleotides fragment was determined. Neither the amino acid composition analysis nor the amino acid sequence derived from the cDNA nucleotide sequence revealed differences between schizophrenics and controls. Thus, no evidence was found for changes in the amino acid sequence of pituitary gamma-endorphin in these analyses, which include 3 cases of schizophrenia.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides. By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35-nucleotide leader sequence (mini-exon) also found on several other trypanosome mRNAs. The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit. The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs. Trypanosome TIM is the most basic of all TIMs sequenced thus far. This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter-containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

A human gastric carcinoma contains a single mutated and an amplified normal allele of the Ki-ras oncogene.

The DNA from various human tumors and tumor cell lines was screened for the presence of mutated ras oncogenes with synthetic oligonucleotide probes, as well as with the NIH/3T3 cell transfection assay. Among the various mutations found we discovered two novel Ki-ras mutations in codon 12: gly to ala and gly to ser. A gastric carcinoma was found to possess a single mutated Ki-ras allele (gly-12 to ser), as well as a 30-50 fold amplified normal allele. This implies that two activating steps must have occurred in this malignancy.

Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia.

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization.

The activation of ras genes in naturally occurring tumors has, thus far, been found to be due to mutations in codon 12 or 61 resulting in single amino acid substitutions. We have used highly labeled synthetic oligonucleotides to detect mutations in these codons and to determine the exact position of the mutation. Using this approach we have found three different mutations in codon 61 of the N-ras gene of various human tumor cell lines. In the fibrosarcoma line HT1080 the first nucleotide of the codon is mutated; in the promyelocytic line HL60 the second and in the rhabdomyosarcoma line RD301 the third nucleotide. For RD301 this implies that the normal glutamine residue at position 61 is replaced by histidine. In addition to the mutated N-ras gene the three cell lines have a normal N-ras gene which is indicative of the dominant character of the mutations.