Evidence for vesicles that mediate long-chain fatty acid uptake by human microvascular endothelial cells

This study analyzes the mechanisms of long-chain fatty acid (LCFA) uptake by human microvascular endothelial cells (HMEC). The time course revealed the presence of an early, carrier-mediated uptake component and a later component mediated by clathrin-coated vesicles (CCV) and caveolae, as evidenced by three different experimental approaches: 1) significant reduction of [3H]oleate uptake over 5 min by either inhibition of CCV formation by potassium depletion or hypertonic medium, or disruption of caveolae by filipin III or cyclodextrin. 2) Co-localization of intracellular 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid with CCV and caveolae using confocal laser scanning microscopy. 3) Enrichment of [3H]oleate in a subcellular fraction containing CCV and caveolae. Within 10 min, more than 75% of intracellular [3H]oleate remained unmetabolized, suggesting that HMEC preferentially shuttle LCFA through the cell using CCV and caveolae as carriers. The uptake of albumin paralleled that of oleate within the first 10 min, suggesting internalization of at least some LCFA bound to albumin. Compared to oleate and albumin, the uptake of sucrose and dextran was low, indicating a potential minor contribution of fluid-phase endocytosis to the total vesicular LCFA uptake. The data indicate a previously unrecognized role of both CCV and caveolae for the uptake of LCFA by HMEC.     

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.     

Effects of a retaining wall and an artificial embankment on nearshore littoral habitats and biota in a large Alpine lake

Hydrobiologia - The littoral zones of many Central European lakes are severely altered by lake-side retaining walls. These are suspected to impair littoral biota due to the reflection of incoming...     

Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Guidelines: From artificial evolution to computational evolution: a research agenda

Computational scientists have developed algorithms inspired by natural evolution for at least 50 years. These algorithms solve optimization and design problems by building solutions that are 'more fit' relative to desired properties. However, the basic assumptions of this approach are outdated. We propose a research programme to develop a new field: computational evolution. This approach will produce algorithms that are based on current understanding of molecular and evolutionary biology and could solve previously unimaginable or intractable computational and biological problems.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Neurons and Neuronal Stem Cells Survive in Glucose-Free Lactate and in High Glucose Cell Culture Medium During Normoxia and Anoxia

Several questions concerning the survival of isolated neurons and neuronal stem and progenitor cells (NPCs) have not been answered in the past: (1) If lactate is discussed as a major physiological substrate of neurons, do neurons and NPCs survive in a glucose-free lactate environment? (2) If elevated levels of glucose are detrimental to neuronal survival during ischemia, do high concentrations of glucose (up to 40 mmol/L) damage neurons and NPCs? (3) Which is the detrimental factor in oxygen glucose deprivation (OGD), lack of oxygen, lack of glucose, or the combination of both? Therefore, in the present study, we exposed rat cortical neurons and NPCs to different concentrations of d-glucose ranging from 0 to 40 mmol/L, or 10 and 20 mmol/L l-lactate under normoxic and anoxic conditions, as well as in OGD. After 24 h, we measured cellular viability by biochemical assays and automated cytochemical morphometry, pH values, bicarbonate, lactate and glucose concentrations in the cell culture media, and caspases activities. We found that (1) neurons and NPCs survived in a glucose-free lactate environment at least up to 24 h, (2) high glucose concentrations >5 mmol/L had no effect on cell viability, and (3) cell viability was reduced in normoxic glucose deprivation to 50% compared to 10 mmol/L glucose, whereas cell viability in OGD did not differ from that in anoxia with lactate which reduced cell viability to 30%. Total caspases activities were increased in the anoxic glucose groups only. Our data indicate that (1) neurons and NPCs can survive with lactate as exclusive metabolic substrate, (2) the viability of isolated neurons and NPCs is not impaired by high glucose concentrations during normoxia or anoxia, and (3) in OGD, low glucose concentrations, but not low oxygen levels are detrimental for neurons and NPCs.