The primary structure of cytochrome c from the nematode Caenorhabditis elegans.

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an 'invariant' phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.     

Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III.

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.     

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed.     

Evidence for reverse cholesterol transport in vivo from liver endothelial cells to parenchymal cells and bile by high-density lipoprotein.

Acetylated low-density lipoprotein (acetyl-LDL), biologically labelled in the cholesterol moiety of cholesteryl oleate, was injected into control and oestrogen-treated rats. The serum clearance, the distribution among the various lipoproteins, the hepatic localization and the biliary secretion of the [3H]cholesterol moiety were determined at various times after injection. In order to monitor the intrahepatic metabolism of the cholesterol esters of acetyl-LDL in vivo, the liver was subdivided into parenchymal, endothelial and Kupffer cells by a low-temperature cell-isolation procedure. In both control and oestrogen-treated rats, acetyl-LDL is rapidly cleared from the circulation, mainly by the liver endothelial cells. Subsequently, the cholesterol esters are hydrolysed, and within 1 h after injection, about 60% of the cell- associated cholesterol is released. The [3H]cholesterol is mainly recovered in the high-density lipoprotein (HDL) range of the serum of control rats, while low levels of radioactivity are detected in serum of oestrogen-treated rats. In control rats cholesterol is transported from endothelial cells to parenchymal cells (reverse cholesterol transport), where it is converted into bile acids and secreted into bile. The data thus provide evidence that HDL can serve as acceptors for cholesterol from endothelial cells in vivo, whereby efficient delivery to the parenchymal cells and bile is assured. In oestrogen-treated rats the radioactivity from the endothelial cells is released with similar kinetics as in control rats. However, only a small percentage of radioactivity is found in the HDL fraction and an increased uptake of radioactivity in Kupffer cells is observed. The secretion of radioactivity into bile is greatly delayed in oestrogen-treated rats. It is concluded that, in the absence of extracellular lipoproteins, endothelial cells can still release cholesterol, although for efficient transport to liver parenchymal cells and bile, HDL is indispensable.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.