Murine toxin of Yersinia pestis shows phospholipase D activity but is not required for virulence in mice.

Purified murine toxin (Ymt) of Yersinia pestis is highly toxic for mice and rats but less active in other animals such as guinea pigs, rabbits, dogs and monkeys. This suggested that Ymt contributes to the very low infectious dose of Y. pestis in mice. The gene encoding Ymt (ymt) is localised on the 100-kb plasmid pFra, which is unique for Y. pestis. Sequence analysis revealed that Ymt showed homology to proteins of the phospholipase D (PLD) superfamily of proteins. Y. pestis strains expressing Ymt possessed PLD activity whereas strains carrying deletions in the ymt gene showed no detectable PLD activity. Western blot analysis showed that Ymt was associated with bacteria under normal growth conditions, and immunogold EM revealed that Ymt was mainly localised in the bacterial cytoplasm. Ymt was purified to homogeneity, and the purified toxin showed a dose-dependent PLD activity. Substitution of amino acids in the PLD consensus motif of Ymt essentially abolished the enzymatic activity and these variants of the toxin were no longer toxic to mice. Interestingly, an in-frame deletion mutant of ymt in the Y pestis strain KIM was not significantly attenuated for mouse virulence. Together with the observation that expression of Ymt was higher at room temperature compared to 37 degrees C this prompted us to investigate the role of Ymt in the flea vector. Fleas were infected with isogenic ymt+ or ymt- mutant strains of Y. pestis. Preliminary results suggest that Ymt is important for survival of Y. pestis in the flea and thereby also for the flea-borne route of infection.

Isolation of a calreticulin-like calcium binding protein from bovine brain.

Intracellular calcium levels are stringently regulated in all cells. The nature of this regulation is incompletely understood, but recent evidence indicates that the endoplasmic reticulum plays an important role in sequestering intracellular calcium. Using methods for isolating both calsequestrin and calreticulin, we have isolated a 58 kDa, high capacity calcium binding protein that exists in microsomes that shift their density in an oxalate-mediated density shift assay. This protein which we call CBP-58 bears similarities to the endoplasmic reticulum protein, calreticulin, in that it has a pI of 4.7 containing approximately 30% glutamate and aspartate, has a high capacity for calcium, and stains blue with the carbocyanine dye, 'Stains-all'. Peptide, amino acid, nucleotide and immunochemical analyses reveal further similarities between CBP-58 and calreticulin, but also some marked differences. Its tissue distribution suggests it is highly enriched in brain versus other tissues. We believe that CBP-58 is a calreticulin-like protein and that differences in the amino acid composition and sequences may reflect species diversity in calreticulin.

Purification and chemical characterization of a cholera toxin-cross-reactive cytolytic enterotoxin produced by a human isolate of Aeromonas hydrophila.

A bacterial protein toxin possessing hemolytic, enterotoxic, and cytotoxic activities as well as cross-reactivity to cholera toxin was purified from culture filtrates of a human diarrheal isolate of Aeromonas hydrophila (SSU). This cytolytic enterotoxin was purified by ammonium sulfate precipitation, hydrophobic chromatography using phenyl-Sepharose, anion-exchange chromatography on DEAE-Bio-Gel A, and size-exclusion high-performance liquid chromatography. The factor was a single polypeptide with an apparent molecular weight of 52,000 as determined by polyacrylamide gel electrophoresis. Automated amino acid sequence analysis confirmed that the toxin was a single chain and established a 25-residue N-terminal segment which was identical to that of aerolysin purified from culture supernatants of A. hydrophila isolate Ah65 originally obtained from rainbow trout as reported by Howard et al. (S. P. Howard, W. J. Garland, M. J. Green, and J. T. Buckley, J. Bacteriol. 169:2869-2871, 1987). However, the amino acid compositional analysis of the toxin produced by our human isolate (SSU) differed significantly from that of the Ah65 isolate. Taken together, these results strongly indicated that several toxic phenomena associated with A. hydrophila (SSU) culture filtrates, including hemolysis, cytotoxicity, and enterotoxicity as well as cross-reactivity to cholera toxin, all can occur on a single polypeptide. In addition, these results underline the fact that although aerolysin-related toxins isolated from culture filtrates of A. hydrophila are biologically similar, significant chemical and immunological differences may exist between toxins produced by individual isolates.

Evidence for production of an enterotoxin and cholera toxin cross-reactive factor by Aeromonas hydrophila.

The enterotoxins produced by Aeromonas hydrophila were examined for biological activity by the rabbit ileal loop and suckling mouse assays, as well as by elongation of CHO cells. Antigenic evaluation of the culture filtrates from various isolates of A. hydrophila was performed by enzyme-linked immunosorbent assay with anti-cholera toxin and anti-Aeromonas enterotoxin. Heat stability data demonstrated the presence of a heat-labile cholera toxin cross-reactive factor and a heat stable non-cholera toxin cross-reactive enterotoxin. The biological activities of both enterotoxins were heat labile at 56 degrees C for 20 min.

Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia.

Three peptide complexes which can induce egg laying in Aplysia were isolated from the atrial gland of the marine mollusc Aplysia californica and chemically characterized. Amino acid sequence analyses established the covalent structures, including disulfide assignments, of all three dimeric complexes. Each complex consisted of an identical 18-residue peptide (A-AP) which was disulfide-bonded to a 36-residue peptide that was homologous to bag cell egg-laying hormone (ELH). The primary structure of A-AP was determined to be: NH2-Asp-Ser-Asp-Val-Ser-Leu-Phe-Asn-Gly-Asp-Leu-Leu-Pro-Asn-Gly-Arg-Cys- Ser-COOH. The primary structure of one of the three ELH-related peptides (A-ELH) was determined to be NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile-Gln-Ala-Arg-Arg-Arg-Cys-Leu-Asp-Ala-Leu-Arg-Gln-Arg-Leu-Leu-Asp- -Leu-COOH. The two other ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH, differed from A-ELH at 1 and 2 residues, respectively. Both [Ala27] A-ELH and [Gln23, Ala27]A-ELH were novel peptide sequences representing products of as yet uncharacterized genes within the ELH family. These structural studies provide the first direct chemical evidence that an 18-residue peptide (A-AP) derived from a polypeptide precursor encoded by the A gene, as predicted from nucleotide sequence analysis, occurs in the atrial gland; the Cys17 residue of A-AP is disulfide-bonded to Cys25 of A-ELH; and A-AP also occurs disulfide-bonded to two additional, previously undescribed ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH.

Isolation of high-specific-activity subunits of cholera toxin by reversed-phase high-performance liquid chromatography.

Facile, rapid procedures for the separation of active cholera toxin subunits were developed, based on high-performance liquid chromatography (HPLC) with a Nucleosil C8 reversed-phase column. These procedures were capable of completely resolving subunits A and B as well as S-carboxymethylated or reduced alpha-, gamma-, and beta-chains. The binding of HPLC-purified B subunit to GM1 ganglioside was essentially identical to that of cholera toxin when compared on a molar basis. The adenosine 5'-diphosphate-ribosyltransferase activity of HPLC-purified A subunit, reduced alpha-chain, or carboxymethyl alpha-chain was also determined to be reasonably high compared to that of cholera toxin or commercially prepared A subunit.

Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor.

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the beta chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of Mr 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the alpha chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by alpha and beta chain regions, respectively.

Prosimian hemoglobins I. The primary structure of the beta-globin chain of Lemur catta.

The primary structure of the beta chain from the hemoglobin of a prosimian primate, Lemur catta, has been determined by automated Edman degradation of intact polypeptide chain and fragments produced by tryptic, cyanogen bromide and acid cleavage, and by homology with the sequence of Lemur fulvus. The sequence presented here differs from the human beta-globin sequence at 26 sites. This is the same degree of divergence previously reported for the beta-globin chain of Lemur fulvus. The sequences of the two congeneric lemuroid beta-globin chains are surprisingly divergent, differing at 18 sites. Of the 26 positions where L. catta differs from Homo sapiens, 7 are at positions with defined function. Of these 7 positions, 4 (2-Phe, 54-Ile, 94-Val, 112-Ile) are unique to L. catta among the primate beta-globin chains of established sequence. Residue 112-Ile is consistent with the prediction of Beard and Goodman (19) of an isoleucyl residue in this position in the ancestral primate beta-globin chain.