Homologous very-long-chain 1,3-alkanediols and 3-hydroxyaldehydes in leaf cuticular waxes of Ricinus communis L

Surface extracts from primary leaves of Castor bean were found to contain 1.8 microg cm(-2) of cuticular waxes. The mixture comprised alkanes (C(26)-C(29)), primary alcohols (C(22)-C(38)), aldehydes (C(26) and C(28)), fatty acids (C(20)-C(34)) and triterpenoids (lupeol, beta- and alpha-amyrin). Besides, a series of n-alkane-1,3-diols was detected, with chain lengths ranging from C(22) to C(28), a strong predominance of even-numbered homologs, and a maximum for hexacosane-1,3-diol. Seven other compounds were assigned to a novel class of wax constituents and identified as homologous unbranched 3-hydroxyaldehydes ranging from C(22) to C(28). As the chain length distribution of this series closely paralleled the homolog pattern of 1,3-diols, it seems likely that both compound classes are biosynthetically related.     

Helicobacter pylori does not mediate the formation of carcinogenic N-nitrosamines

Background. Both N‐nitroso compounds and colonization with Helicobacter pylori represent known risk‐factors for the development of gastric cancer. Endogenous formation of N‐nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N‐nitroso compounds. Based on experiments with a noncarcinogenic N‐nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N‐nitrosamines. Materials and Methods. Bacteria were grown in the presence of 0–1000 µM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3. Results. Incubation of Neisseria cinerea (positive control) with 500 µM morpholine and 500 µM nitrite, resulted in a significant increase in formation of N‐nitrosomorpholine, but there was no significant induction of N‐nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions. Conclusion. H. pylori does not induce formation of the carcinogenic N‐nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficultly nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.     

Gibberellins in seeds of Arabidopsis thaliana: biological activities,identification and effects of light and chilling on endogenous levels

The activities of several gibberellins in stimulating germination of wild-type and GA-deficient gal seeds of Arabidopsis thaliana were compared. Of the six compounds tested GA₄ and GA₇-isolactone had the highest activity and GA₇ and GA₉ the lowest; activities of GA₁ and GA₃ were intermediate. Combined application of pure GAs presented no indications that more than one GA receptor is involved. Four GAs were identified in extracts from wild-type and GA-insensitive gai seeds by combined gas chromatography mass spectrometry: GA₁, GA₃, GA₄ and GA₉. Effects of light and chilling on levels of GA₁, GA₄ and GA₉ were studied using deuterated standards. Light increased both GA levels and germination in unchilled wild-type and gai seeds. As a result of irradiation GA levels in gai seeds were 7–10 times as high as in wild-type seeds. In the dark germination was 0%, in the light 14% of gai seeds and 95% of wild-type seeds germinated. A chilling pre-treatment of 7 days at 2°C was required to enhance further the germination of gai seeds in the light. Light did not increase GA levels of chilled seeds of either genotype; levels of GA₄ and GA₉ of chilled gai seeds, in the light were respectively 7 and 12 times lower than in non-chilled seeds, whereas the latter seeds germinated better. Slightly elevated levels of GA₄ were detected in darkness after chilling, but germination capacity was still 0%. These results strengthened the conclusion that GAs are required for germination of A. thaliana seeds, whereby GA₄ has intrinsic biological activity. However, it is unlikely that light and chilling stimulate germination primarily by increasing levels of GA. Instead GA sensitivity is a possible alternative.     

Differential lipoprotein transport pathways of K-vitamins in healthy subjects

Vitamin K is a group name for K1 (phylloquinone) and K2 (menaquinones). Both forms contribute to the tissue vitamin K status. Following intestinal absorption, the serum transport of these lipophilic compounds to their target tissues takes place via lipoproteins. In previous studies we have found that K1 is preferentially accumulated in the liver, whereas menaquinones have a more widespread distribution pattern. Here we have tested whether these differences may be explained by the different liposolubility of the various K-vitamers, resulting in their association with different lipoprotein particles. Six healthy male volunteers received a mixture containing 2 micromol of each of three K vitamers (K1, MK-4, and MK-9) dissolved in corn oil. Blood was obtained at baseline and at different time intervals after intake for the measurement of vitamin K in serum and in the lipoprotein fractions. During the first 4 h after intake all K-vitamins were found to be associated predominantly with the triacylglycerol-rich lipoprotein (TGRLP) fraction. Since the TGRLP fraction is mainly cleared by the liver, this suggests that initially most of the K-vitamins are transported to the liver. In contrast to K1, however, both menaquinones investigated were also found in TGRLP and low-density lipoprotein, whereas MK-4 was even present in high-density lipoprotein. This explains why menaquinones may have a different distribution profile and suggests a relatively large impact of menaquinones on extra-hepatic vitamin K status than generally assumed. Moreover, the very long half-life time of MK-9 in the circulation indicates that it may form a more constant source of vitamin K than are either K1 or MK-4.     

Heat stress activates phospholipase D and triggers PIP2 accumulation at the plasma membrane and nucleus

Heat stress induces an array of physiological adjustments that facilitate continued homeostasis and survival during periods of elevated temperatures. Here, we report that within minutes of a sudden temperature increase, plants deploy specific phospholipids to specific intracellular locations: phospholipase D (PLD) and a phosphatidylinositolphosphate kinase (PIPK) are activated, and phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP₂) rapidly accumulate, with the heat-induced PIP₂ localized to the plasma membrane, nuclear envelope, nucleolus and punctate cytoplasmic structures. Increases in the steady-state levels of PA and PIP₂ occur within several minutes of temperature increases from ambient levels of 20–25°C to 35°C and above. Similar patterns were observed in heat-stressed Arabidopsis seedlings and rice leaves. The PA that accumulates in response to temperature increases results in large part from the activation of PLD rather than the sequential action of phospholipase C and diacylglycerol kinase, the alternative pathway used to produce this lipid. Pulse-labelling analysis revealed that the PIP₂ response is due to the activation of a PIPK rather than inhibition of a lipase or a PIP₂ phosphatase. Inhibitor experiments suggest that the PIP₂ response requires signalling through a G-protein, as aluminium fluoride blocks heat-induced PIP₂ increases. These results are discussed in the context of the diverse cellular roles played by PIP₂ and PA, including regulation of ion channels and the cytoskeleton.     

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4⁺ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC₅₀ of 17.38 versus 22.01 μM for cucurbitacin E and 13.36 versus 24.47 μM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC₅₀ of 9.98 and 29.15 μM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.     

Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with <Superscript>2</Superscript>H NMR experiments

Order parameters from deuterium NMR are often used to validate or calibrate molecular dynamics simulations. This paper gives a short overview of the literature in which experimental order parameters from ²H NMR are compared to those calculated from MD simulations. The different ways in which order parameters from experiment are used to calibrate and validate simulations are reviewed. In the second part of this review, a case study of cholesterol in a DMPC bilayer is presented. It is concluded that the agreement between experimental data and simulation is favorable in the hydrophobic region of the membrane, for both the phospholipids and cholesterol. In the interfacial region the agreement is less satisfactory, probably because of the high polarity of this region which makes the correct computation of the electrostatics more complex. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.