Apolipoprotein A-I-containing lipoproteins with pre-beta electrophoretic mobility

Among the apoA-I-containing lipoproteins isolated by selected-affinity immunosorption from human serum and plasma, we have identified a subpopulation which, unlike the bulk of high density lipoproteins, has pre-beta electrophoretic mobility. This pre-beta subpopulation can be observed directly in fresh plasma by immunoelectrophoresis. It contains phospholipid and free and esterified cholesterol, but protein constitutes 90% of its mass. Apolipoprotein A-I is the predominant apolipoprotein in this subpopulation; apolipoprotein A-II and the B lipoproteins are not detected. The protein moiety of this subpopulation exhibits markedly lower helicity than that of high density lipoproteins isolated by ultracentrifugation.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

ARE PARALLEL MORPHOLOGIES OF CAVE ORGANISMS THE RESULT OF SIMILAR SELECTION PRESSURES?

The amphipod Gammarus minus is present in both caves and springs, with cave populations showing elaborated (size and antennae) and reduced (eye) characters relative to spring populations. Earlier studies have shown that cave populations resulted from independent invasions of hydrologically isolated subterranean drainages and that there is genetic variation for both elaborated and reduced characters. In this study we tested the hypothesis that a similar pattern of selection on isolated cave populations is responsible for the parallel evolution of cave morphologies. We used variation in mating success and fecundity to test for the presence of directional selection on eye, antennal, and body size characters in a set of cave and spring populations during a series of seasonal cross-sectional samplings. We found significant directional selection for smaller eyes in caves and larger eyes in springs, which supports the hypothesis that selection is responsible for reduced eye size in cave populations. We also found selection for larger body and antennal size in cave populations, which is consistent with the hypothesis that parallel patterns of selection in caves are responsible for the elaboration of body and antennal size. However, we found selection for larger body and antennal size in spring populations that is not consistent with the observed divergence of spring and cave populations. We suggest that unmeasured components of viability selection could be more important in springs than in caves and may act against the selection for larger size found in spring populations.     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia.

Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.