Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

Replication and discrimination of limb movement velocity

It is well known that proprioception is composed of the senses of movement and position. Whereas tests of position sense are quite commonly used, tests of the acuity in perception of movement velocity are scarce. In the present study we examined some novel tests for assessing the sense of limb movement velocity, involving replication and discrimination of single-joint movement velocity. Specifically, we investigated: (1) whether replication of limb movement velocity is more accurate following active criterion movements as compared to passive; (2) whether antagonist muscle contraction during passive limb movement enhances velocity discrimination; (3) how criterion movement velocity influences response accuracy; (4) the relationship between movement velocity and movement extent during velocity replication; and (5) whether subjects really base discrimination of velocities on perceived velocity. Sixteen healthy subjects participated in four tests (I-IV). For each test, horizontal abductions were performed about the right glenohumeral joint from the sagittal plane. The subjects were required to actively replicate the velocity of either an active (Test I) or passive (Test II) criterion movement, or judge whether a passive/semipassive (passive during antagonist muscle contraction) movement was faster or slower than a previous passive/semipassive criterion movement (Test III/IV). The results revealed higher response accuracy for Test I compared to Test II and for slower movements compared to faster, but no difference in response accuracy between Test III and IV. For velocity discrimination, the analysis revealed that the subjects based their judgment on the difference between criterion and comparison velocity rather than time or extent cues.     

Cell transduction pathways of transportans

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.     

Evolution of specialization in resource utilization in structured metapopulations

We study the evolution of resource utilization in a structured discrete-time metapopulation model with an infinite number of patches, prone to local catastrophes. The consumer faces a trade-off in the abilities to consume two resources available in different amounts in each patch. We analyse how the evolution of specialization in the utilization of the resources is affected by different ecological factors: migration, local growth, local catastrophes, forms of the trade-off and distribution of the resources in the patches. Our modelling approach offers a natural way to include more than two patch types into the models. This has not been usually possible in the previous spatially heterogeneous models focusing on the evolution of specialization.     

Carbohydrate receptor specificity of K99 fimbriae of enterotoxigenicEscherichia coli

K99 Fimbriae from enterotoxigenicEscherichia coli (ETEC) were found to bind specifically to sialic acid, as measured in a haemagglutination inhibition assay using the intact bacteria and human erythrocytes. The affinity forN-glycolylneuraminic acid was about twice that ofN-acetylneuraminic acid (NeuAc), and other monosaccharides were found to be at least ten-fold less effective as inhibitors. The specificity was found to depend on electrostatic interaction where the carboxyl group and its orientation plays an important role. 2--Benzyl-NeuAc was a better inhibitor than 2--methyl-NeuAc suggesting a hydrophobic patch near the binding site on the protein. Axially oriented hydroxyl groups as in 4-epi-NeuAc and 3-hydroxy-NeuAc seemed to participate in binding since these derivatives were better inhibitors thanN-acetylneuraminic acid. K99 was found to have a higher affinity for 4-O-acetyl-NeuAc and lower affinity forN-acetylneuraminic acid withO-substituents at C7-C9 as compared toN-acetylneuraminic acid. Hence, the degree ofO-acetylation of sialic acid in the mucosa of the small intestine may influence colonization and determine susceptibility to infection.