Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet

To investigate the factors governing the accumulation of sucroseand amino acids in the taproots of sugar beet, their contentswere measured in the leaves, phloem sap and the taproots ofsugar beet, fodder beet and a hybrid between both, grown oneither 3.0 or 0.5 mM nitrate. In the taproots the contents ofmalate, citrate and inorganic ions were also determined. Forthe high sucrose accumulation in sugar beet as compared to theother varieties three factors were found. (a) In sugar beet,less amino acids and more sucrose are taken up into the phloemthan in fodder beet. (b) In sugar beet, the sucrose and aminoacid syntheses are less sensitive to the nitrate concentrationsthat are required for optimal plant growth than in other varieties.In fodder beet, upon raising the nitrate concentration from0.5 mM to 3 mM, the synthesis and storage of sucrose is decreasedand that of amino acids increased. The corresponding valuesin sugar beet (0.5 mM) are similar to those in fodder beet andare not much affected by an increase of nitrate. (c) The sucroseaccumulation is limited by the accumulation of inorganic ionsin the taproots. The sucrose content in the taproots is negativelycorrelated to the total ion content. Whereas sucrose representstwo-third of all solutes in the taproots of sugar beet, it amountsto only one-third of the solutes in fodder beet taproots. Key words: Amino acids, Beta vulgans L, phloem sap, potassium, sucrose storage, sugar beet, taproots, transport     

Molekularbiologie der Mukoviszidose

Mukoviszidose ist eine angeborene Funktionsstörung der exokrinen Körperdrüsen. Sie kommt bei etwa 1 : 2500 Lebendgeborenen in der weißen Bevölkerung Europas und Nordamerikas vor und gehört damit zu den häufigsten lebensbedrohlichen Erbkrankheiten des Menschen. Dennoch sind vollständige Beschreibungen des Krankheitsbildes erst seit etwas mehr als fünfzig Jahren bekannt. Die bei den meisten Mukoviszidosekranken auftretenden fibrotischen Veränderungen (Fibrose - Vermehrung des Bindegewebes) der Bauchspeicheldrüse haben zu der Bezeichnung cystic fibrosis of the pancreas geführt, so daß auch der Name zystische Fibrose (cystic fibrosis, CI) heute synonym gebräuchlich ist. Wir wollen im folgenden in kurzer Form auf die Symptome und Ursachen der Erkrankung eingehen. Eine ausführlichere Darstellung der Mukoviszidose und ihrer molekularbiologischen Grundlagen mit zahlreichen Literaturhinweisen ist kürzlich aktualisiert und erweitert worden [21].     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Endosome-lysosome fusion at low temperature

Based on an initial study (Dunn, W. A., Hubbard, A. L., and Aronson, Jr., N. N. (1980) J. Biol. Chem. 255, 5971-5978), low temperature is often used to selectively inhibit fusion between endosomes and lysosomes. Here we have tried to characterize the nature of this inhibition. In addition to endocytic contents markers, we have used a covalent membrane marker to measure the interaction between endosomes and lysosomes over extended periods of time at low temperature. Mouse macrophage cells (P388D1) and human skin fibroblasts were enzymatically labeled with radioactive galactose to provide a covalent marker for plasma-membrane glycoconjugates. Subsequent endocytic membrane traffic for 24 h at 16 degrees C resulted in a significant transfer of membrane marker, as well as of endocytic contents marker, to high density lysosomes, as observed by subcellular fractionation. The kinetics of this transfer have been analyzed for macrophages using the membrane marker, horseradish peroxidase as fluid-phase, and iodinated acetyl low density lipoprotein as receptor-mediated endocytic contents marker. Transfer to lysosomes occurred only about 6 h after application of the respective marker at 16 degrees C. When transfer to lysosomes was initiated by 15 min preincubation at 37 degrees C, subsequent cooling to 16 degrees C did not inhibit ongoing transfer which continued with the same kinetics as when observed after the lag phase. These results show that low temperature delays an unidentified pre-fusion step, but does not inhibit endosome-lysosome fusion as such.     

The metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.Hepatocellular carcinoma (HCC)¹ currently is the fifth most common malignancy worldwide with an annual incidence up to 500 per 100,000 individuals depending on the geographic region investigated. Whereas 80% of new cases occur in developing countries, the incidence increases in industrialized nations including Western Europe, Japan, and the United States (1). To manage patients with HCC, tumor markers are very important tools for diagnosis, indicators of disease progression, outcome prediction, and evaluation of treatment efficacy. Several tumor markers have been reported for HCC, including α-fetoprotein (AFP) (2), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) (3), and des-γ-carboxyl prothrombin (DCP) (4). However, none of these tumor markers show 100% sensitivity or specificity, which calls for new and better biomarkers.To identify novel biomarkers of HCC, many clinical studies using “omics”-based methods have been reported over the past decade (5–6). In particular, the proteomics-based approach has turned out to be a promising one, offering several quantification techniques to reveal differences in protein expression that are caused by a particular disease. In most studies, the well-established 2D-DIGE technique has been applied for protein quantification followed by identification via mass spectrometry (7–15). Even if the quantification is very accurate and sensitive in this gel-based approach, the relatively high amount of protein sample necessary for protein identification is the major disadvantage of this technique. Several mass-spectrometry-based quantitative studies using labeling-techniques like SILAC (stable isotope labeling by amino acids in cell culture) or iTRAQ (isobaric tags for relative and absolute quantification) have also been carried out for biomarker discovery of HCC (16–18). Here, the concomitant protein quantification and identification in a mass spectrometer allows high-throughput analyses. However, such experiments imply additional labeling reactions (in case of iTRAQ) or are limited to tissue culture systems (in case of SILAC). In the latter case, one can overcome the limitation by using the isotope-labeled proteins obtained from tissue culture as an internal standard added to a corresponding tissue sample. This approach is known as CDIT (culture-derived isotope tags) and was applied in a HCC study, very recently (19). Label-free proteomics approaches based on quantification by ion-intensities or spectral counting offer another possibility for biomarker discovery. These approaches are relatively cheap compared with the labeling approaches, because they do not require any labeling reagents and furthermore they allow for high-throughput and sensitive analyses in a mass spectrometer. A quantitative study of HCC using spectral counting has been reported (20), whereas to our knowledge an ion-intensity-based study has not been performed yet. Apart from these quantification strategies, protein alterations in HCC have been studied by MALDI imaging, as well. Here, the authors could show that based on its proteomic signature, hepatocellular carcinoma can be discriminated with high accuracy from liver metastasis samples or other cancer types (21) as well as liver cirrhosis (22). Based on these results, it could be assumed that MALDI imaging might be a promising alternative to standard histological methods in the future.Here, we report a quantitative proteomic study that combines two different techniques, namely the well-established 2D-DIGE approach and a label-free ion-intensity-based quantification via mass spectrometry and liquid chromatography. To our knowledge this is the first time such a combined study was performed with regard to hepatocellular carcinoma. By comparing the results of both studies, we aim to identify high-confident biomarker candidates of HCC, as gel- and LC-MS-based techniques are complementary. To verify the differential protein expressions detected in our proteomic studies we performed additional immunological verifications for selected proteins within two different sample sets (Fig. 1).Open in a separate windowFig. 1.Schematic representation of the applied workflow.