Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes

The gene cluster that codes for feedback-resistant aspartate kinase (lysCα and lysCβ) and aspartate semialdehyde dehydrogenase (asd) was cloned from a mutant strain of Corynebacterium glutamicum. Its functional analysis by subcloning, enzyme assays, and type of aspartate kinase regulation enabled the isolation of a fragment for separate expression of the feedback-resistant kinase without aspartate semialdehyde dehydrogenase expression. This was used together with other clones constructed (J. Cremer, L. Eggeling, and H. Sahm, Mol. Gen. Genet. 220:478-480, 1990) to overexpress individually each of the six genes that convert aspartate to lysine. Analysis of lysine formation revealed that overexpression of the feedback-resistant kinase alone suffices to achieve lysine formation (38 mM). Also, sole overexpression of wild-type dihydrodipicolinate synthase resulted in lysine formation but in a lower amount (11 mM). The other four enzymes had no effect on lysine secretion. With a plasmid overexpressing both relevant enzymes together, a further increase in lysine yield was obtained. This shows that of the six enzymes that convert aspartate to lysine the kinase and the synthase are responsible for flow control in the wild-type background and can be useful for construction of lysine-producing strains.     

Degradation of coniferyl alcohol and other lignin-related aromatic compounds by Nocardia sp. DSM 1069

Nocardia sp. DSM 1069 was grown on mineral salt media with coniferyl alcohol, 4-methoxybenzoic acid, 3-methoxybenzoic acid or veratric acid as sole sources of carbon and energy. During incubation on coniferyl alcohol, the formation of coniferyl aldehyde, ferulic acid and quantitative accumulation of vanillic acid and proteocatechuic acid could be achieved with mutants. Washed cell suspensions of N. sp. grown on 4-methoxybenzoic acid, oxidized 4-hydroxybenzoic acid and protocatechuic acid. Cells grown on veratric acid, oxidized vanillic acid, isovanillic acid, and protocatechuic acid. Cell extracts were shown to cleave protocatechuic acid by ortho-fission.A mutant without protocatechuate 3,4-dioxygenase activity was not influenced in its growth on 3 methoxybenzoic acid. Cell free extracts of cells grown on 3-methoxybenzoic acid were shown to catalyze the oxidation of gentisic acid (2,5-dihydroxybenzoic acid). The resulting ring cleavage product was further metabolized by a glutathione dependent reaction.The specificity of the demethylation reactions has been investigated with a mutant unable to grow on vanillic acid. This mutant was not impaired in the degradation of isovanillic acid, 4-methoxy-, or 3-methoxybenzoic acid, whereas growth of this mutant on veratric acid (3,4-dimethoxybenzoic acid) was only half as much as that of the wild type. Concomitantly with growth on veratric acid this mutant accumulated vanillic acid with a yield of about 50%.A pathway for the catabolism of coniferyl alcohol, involving oxidation and shortening of the side chain, and of 4-methoxybenzoic acid and veratric acid with protocatechuic acid as intermediate is being proposed. A second one is proposed for the degradation of 3-methoxybenzoic acid with gentisic acid as intermediate.     

Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.