Carbonates, thiocarbonates, and the corresponding monoalkyl derivatives. 1. Their preparation and isotropic (13)C NMR chemical shifts.

Three series of potassium carbonate and thiocarbonate salts were synthesized, and the corresponding (13)C isotropic solid-state NMR and the aqueous solution (13)C and (1)H NMR data were collected. The series of compounds that were studied consists of (1) the parent compounds, i.e., potassium carbonate, K(2)CO(3), potassium hydrogen carbonate, KHCO(3), potassium monothiocarbonate, K(2)CO(2)S, potassium dithiocarbonate, K(2)COS(2), and potassium trithiocarbonate, K(2)CS(3), (2) the oxygen monoalkyl substituted derivatives of the parent compounds (OR series), i.e., three potassium O-alkylcarbonates, KO(2)COR, three potassium O-alkylmonothiocarbonates, KOSCOR, and three potassium O-alkyldithiocarbonates, KS(2)COR, all with R = CH(3), CH(2)CH(3), CH(CH(3))(2), and (3) the sulfur monoalkyl substituted derivatives of the parent compounds (SR series), i.e., two potassium S-alkylmonothiocarbonates, KO(2)CSR; two potassium S-alkyldithiocarbonates, KOSCSR, and two potassium S-alkyltrithiocarbonates, KS(2)CSR, all with R = CH(3) or CH(2)CH(3). The preparation and proper characterization of KO(2)CSR and KOSCSR with R = CH(3) and CH(2)CH(3) along with new IR and X-ray powder diffraction data for several other compounds in the series are reported for the first time in this study. Solution NMR data for KO(2)CSR (R = CH(3), CH(2)CH(3)) and KOSCSR (R = CH(3)) and solid-state NMR data for K(2)CO(2)S and K(2)COS(2) could not be obtained because they are unstable under the corresponding measurement conditions. The isotropic chemical shift values of the central carbon atoms obtained from solid-state MAS (magic angle spinning) NMR experiments deviate at most by 3 ppm from the corresponding solution values. Two major trends in the (13)C chemical shift values of the central carbon atoms were found. First, if an oxygen atom in a parent compound or in an alkyl-substituted derivative is replaced by a sulfur atom, a significantly higher chemical shift value is observed. This trend is discussed in terms of the paramagnetic contribution to the chemical shielding constant. Second, the size of the alkyl group in the monoalkyl derivatives has a very small effect on the chemical shift values of the central carbon atoms. This observation is explained using the concept of varying inductive effects produced by alkyl groups. The trends observed for the (13)C and (1)H chemical shift values of the alkyl groups follow common concepts on the structure dependency of chemical shifts.

Carbonates, thiocarbonates, and the corresponding monoalkyl derivatives. 2. X-ray crystal structure of potassium methyltrithiocarbonate (KS(2)CSCH(3)).

This study presents the first crystal structure determination of a potassium S-alkylthiocarbonate, the title compound potassium methyltrithiocarbonate (KS(2)CSCH(3)) Single crystals of KS(2)CSCH(3) were obtained by the slow introduction of methylene chloride into a saturated solution of KS(2)CSCH(3) in a 1:1 mixture of methylene chloride and tetrahydrofuran at 0 degrees C in a dry N(2) atmosphere. The compound crystallizes in the monoclinic space group P2(1)/c containing Z = 4 K(+) cations and S(2)CSCH(3-) anions per unit cell. The unit cell dimensions are a = 7.6639(3) A, b = 6.5804(2) A, c = 12.8426(5) A, and beta = 91.565(2) degrees. The isomorphism to the structurally closely related compounds KO(2)COCH(3), KOSCOCH(3), and KS(2)COCH(3) is examined.

Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2.

Mice and rabbits immunized with recombinant forms of malaria vaccine candidate antigens rhoptry-associated proteins 1 and 2 (RAP-1, RAP-2 and rRAP-1, rRAP-2) produce antibodies at titres equivalent to monoclonal antibody ascites fluid raised against the native proteins. Sera from animals immunized with rRAP-1 contain antibodies which recognize the native protein by indirect immunofluorescence and immunoblotting, partially inhibit erythrocyte invasion in vitro and are long lasting. Epitope mapping shows these antibodies predominantly recognize epitopes in the N-terminal third of rRAP-1, some of which coincide with the targets of inhibitory monoclonal antibodies. By contrast, sera from animals immunized with rRAP-2 contain antibodies which recognize the recombinant but not the native protein.

Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors.

To probe the ligand receptor interface, a number of point mutations were introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis. The mutated proteins were expressed in Escherichia coli and analyzed for selective binding to recombinant 55- and 75-kDa TNF receptors in competition with radiolabeled wild-type TNF alpha. Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, whereas mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor. Mutation of the conserved Tyr87 resulted in a dramatic loss of binding activity to both receptors. The selectivity for one or the other receptor type was found to be enhanced by combining two or three point mutations, the effects of the single mutations with respect to receptor selectivity being at least additive. A combination of the mutations Arg32-->Trp and Ser86-->Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor. In contrast, combining the Asp143-->Asn and Ala145-->Arg mutations (D143N-A145R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-fold. In functional assays, selective activation of the 55-kDa TNF receptor by the R32W-S86T mutant elicited a full cytotoxic response in human KYM-1 cells and secretion of interleukin 6 and granulocyte-macrophate colony-stimulating factor in human umbilical vein endothelial cells. In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A145R mutant as well as with agonistic antibodies failed to induce these responses.

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55.

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF-R75, and by receptor type-specific agonists, binding exclusively to TNF-R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha-dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF-R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences.

Ability of recombinant or native proteins to protect monkeys against heterologous challenge with Plasmodium falciparum.

To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively high conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gp190 and its processed products. All antigens were derived from P. falciparum K1, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the K1 sequence, essentially the MAD-20 allelic form of gp190. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gp190, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gp190. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gp190 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity.

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.

Determinants for protein translocation across mammalian endoplasmic reticulum. Membrane insertion of truncated and full-length prelysozyme molecules.

The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full-length prelysozyme using transcription-coupled capping of RNA and subsequent translation in a wheat germ cell-free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal-recognition particles (SRP) to the cell-free system blocked the nascent chain synthesis. The SRP-depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP-induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum.

A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription-coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine-labelled proteins produced offer a number of possibilities for the study of structure-function relationships of proteins.

Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes.

The copy number of plasmids containing the ColE1 replicon is affected by changes in the transcriptional activity within the plasmid if these changes lead to transcriptional readthrough into the replication region towards the promoter priming DNA replication. Such readthrough e.g., from the tet region in pBR322 not only causes overproduction of a peptide known to affect the copy number negatively but also appears to interfere negatively with the replication of the plasmid itself. The proper placement of efficient transcriptional terminators prevents such interference and permits the stable integration of strong promoters. Due to this termination effect, up to 9-fold differences in plasmid copy number were observed, depending upon the particular growth conditions. The higher copy number is of course reflected by higher yields of plasmid-specified gene products indicating the relevance of the above effects for studies of gene expression.