Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Structural analysis of an HLA-B27 population variant, B27f. Multiple patterns of amino acid changes within a single polypeptide segment generate polymorphism in HLA-B27

The structure of a new HLA-B27 variant, B27f, distinguishable from other HLA-B27 subtypes by isoelectric focusing and serologic criteria, has been established by comparative peptide mapping and radiochemical sequence analysis. HLA-B27f differs from the major B27.1 subtype in three clustered amino acid replacements: Asp74, Asp77, and Leu81 in B27.1 are changed to Tyr74, Asn77, and Ala81, respectively in B27f. This pattern of differences is analogous to that of HLA-B27.2 in that this subtype also differs from B27.1 in multiple clustered substitutions within the same segment. Thus, polymorphism within the HLA-B27 system is being achieved by introducing different sets of amino acid changes within a particular short segment of the alpha 1 domain. The most likely mechanism for the introduction of multiple changes within this segment is a nonreciprocal recombination event, such as gene conversion. The structural analogies and ethnic distribution of B27f and B27.2 as compared with those of B27.3, and B27.4 support a dynamic model of HLA-B27 evolution in which polymorphism has been created after the separation of the major ethnic groups. In this model, a Caucasian branch would be characterized by subtypes differing from B27.1 in a few changes within the alpha 1 domain, which were probably generated by single genetic steps. An Oriental branch would include those subtypes which differ from B27.1 by changes in both alpha 1 and alpha 2, involving multiple genetic steps for their generation.     

Sialic acid is a cell surface component of Entamoeba invadens trophozoites

The surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.     

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Epidemiology of tegumentary leishmaniasis in Mérida, Venezuela. I. Diversity and dispersion of phlebotomine species at 3 altitude levels and its possible role in the transmission of the disease

As part of an epidemiological study on leishmaniasis in Merida, Venezuela, the diversity and dispersion of sandflies species found in 15 localities between 175 m and 1,960 m.a.s.l., are presented. From 7,126 collected sandflies (5,132 female and 1,994 male), 24 species were identified, 10 of them recognized as anthropophilic. The relation species-altitude is presented, and the species composition found in human dwellings, periodomestic and sylvatic areas, are recorded. The possible role of the identified species on the transmission of leishmaniasis in the andean region, is discussed.     

Molecular analysis of HLA-A2.4 functional variant KLO: close structural and evolutionary relatedness to the HLA-A2.2 subtype

The structure of an HLA-A2.4 functional variant (A2.4c) expressed on donor KLO has been examined by comparative peptide mapping with other HLA-A2 antigens of known structure and radiochemical sequencing. All the peptide differences between A2.4c and A2.1 could be accounted for by five amino acid changes at positions 9, 43, 66, 95, and 156. The nature of residues 9, 43, and 95 in A2.4c was determined by sequencing to be identical to those in A2.2Y. The nature of residue 156 in A2.4c was also assigned as identical to that in A2.2Y on the basis of the identity of the corresponding peptide in its chromatographic comparison with A2.2Y. Position 66 was unique to A2.4c. It was determined to be an Asn residue instead of the Lys present in all other HLA-A2 antigens of known structure. This was the only detected amino acid difference between A2.4c and A2.2Y. The results indicate that, from a structural point of view, A2.4c is most closely related to the A2.2 subtype antigens and not to other A2.4 antigens. The data are compatible with the assumption that A2.4c was derived from A2.2Y by a single point mutation event.     

An HLA-A2 population variant with structural polymorphism in the α3 region

The HLA-A2 antigen expressed by donor OZB can be distinguished from the main HLA-A2.1 subtype by isoelectric focusing - it is one charge unit more acidic — and by some alloreactive T-cell clones but not by cytolytic T lymphocyte lines. The structure of variant OZB has been examined by comparative peptide mapping with A2.1 and radiochemical sequence analysis. The two molecules were found to differ in a single tryptic peptide from the 0 region, spanning residues 220–243. The amino acid sequence of this peptide from variant OZB revealed that there was only one amino acid change of Glu instead of Ala at position 236, a hitherto invariant residue in class I HLA antigens. All previously characterized HLA or H-2 natural variants have structural changes restricted to the 1 and/or 2 domains. Thus, variant OZB is unique in that (1) it has one amino acid change in 3 and (2) it has no changes in l and 2. The only detected substitution of this variant may be accounted for by a single base change at the DNA level, suggesting that it might have resulted from a point mutation in the A2.1 gene. The structural features of variant OZB open a novel way to examine the influence of polymorphism in 3 on cytolytic T-cell recognition of naturally occurring class I antigens.Abbreviations CTL cytolytic T lymphocytes - HPLC high performance liquid chromatography - IEF isoelectric focusing - MHC major histocompatibility complex