A biocatalyst for the removal of sulfite from alcoholic beverages

The presence of sulfites in alcoholic beverages, particularly in wines, can cause allergic responses with symptoms ranging from mild gastrointestinal problems to life threatening anaphylactic shock in a substantial portion of the population. We have developed a simple and inexpensive biocatalytic method that employs wheatgrass (Triticum aestivum) chloroplasts for the efficient oxidation of sulfites in wines to innocuous sulfates. A sufficiently high rate of sulfite oxidation was obtained in the presence of ethanol at concentrations commonly found in most wines. Crude chloroplast preparations at a concentration as low as 5 mg/mL were capable of reducing sulfite in commercial white wines from 150 ppm to under 7.5 ppm within 3 hours. A 93% removal of sulfite in commercial red wines was observed with 1 mg/mL chloroplasts within 45 min. Optimal sulfite removal efficiency was observed at pH 8.5 and was promoted by illumination, indicating the participation of light-induced photosynthetic electron transport processes in sulfite oxidation. Overall, this work indicates that biocatalytic oxidation using wheatgrass chloroplasts can be employed to remove sulfites from beverages prior to consumption.     

Laminin-1 is phosphorylated by ecto-protein kinases of monocytes

Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties. In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([gamma-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS-PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls. These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes.     

Preparative expression of secreted proteins in bacteria: status report and future prospects

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.     

Positive selection for loss-of-function tat mutations identifies critical residues required for TatA activity

The Tat system, found in the cytoplasmic membrane of many bacteria, is a general export pathway for folded proteins. Here we describe the development of a method, based on the transport of chloramphenicol acetyltransferase, that allows positive selection of mutants defective in Tat function. We have demonstrated the utility of this method by selecting novel loss-of-function alleles of tatA from a pool of random tatA mutations. Most of the mutations that were isolated fall in the amphipathic region of TatA, emphasizing the pivotal role that this part of the protein plays in TatA function.     

beta-Carotene production and its role in sclerotial differentiation of Sclerotium rolfsii

The fungus Sclerotium rolfsii produces beta-carotene, the main detected carotenoid, in levels dependent upon oxidative growth conditions and upon differentiation. beta-Carotene accumulation is 5-, 6.5-, and 6.7-fold higher in undifferentiated mycelia, sclerotia, and differentiated mycelia, respectively, at high than at low oxidative stress. It accumulates more in older than in younger mycelia and is 2-fold higher in differentiated than in undifferentiated mycelia. We propose that beta-carotene is formed possibly to help the fungus reduce oxidative stress that develops during growth. This is supported by the finding that exogenous beta-carotene at non-growth-inhibiting concentrations causes a concentration-dependent reduction of oxidative stress (lipid peroxidation) of undifferentiated mycelia, which results in an equally proportional reduction of sclerotial differentiation. The data of this study support our hypothesis that sclerotial differentiation is induced by oxidative stress.     

Enhanced UV-B radiation,flower attributes and pollinator behaviour in Cistus creticus: a Mediterranean field study

The aim of this investigation was to examine the reasons for the higher pollination success in Cistus creticus under enhanced UV-B radiation (Stephanou & Manetas 1998). Thus, a selected array of floral attributes as well as the frequency and duration of insect visits were studied in the field under ambient or ambient plus supplemental UV-B radiation, simulating a 15% ozone depletion over Patras (38.3° N, 29.1° E). Video-recording revealed two categories of visitors, i.e. true pollinators (bees) and nectar thieves. The frequency of visits to both control and UV-B treated plants was the same and independent of whether the UV-B tubes were on or off during video recording. UV-B radiation had no effect on gross floral morphology (petal surface area, number of pollen grains, stamens and ovules, optical properties of petals and stamens), yet nectary size was almost doubled. In addition, the duration of insect visits was significantly longer on UV-B treated plants, provided that the UV-B tubes were off during monitoring. The differences were abolished during the part of the day that the tubes were on, indicating that the insects were annoyed by supplemental UV-B radiation. These results are consistent with the nectaries producing larger quantities of nectar, which caused the insects to stay longer on flowers of UV-B treated plants and improved pollination success.     

Engineering antibody fragments to fold in the absence of disulfide bonds

Disulfide bonds play a critical role in the stabilization of the immunoglobulin β-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.     

DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function

Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.Death-associated protein kinase (DAPK) 2 shares a high level of homology within its kinase domain with the other two DAPK family members, DAPK1 (DAPk) and DAPK3 (ZIPK/DLK). Since the identification of DAPK1 by Kimchi and co-workers¹ numerous studies have shown that DAPK1 functions as a tumour suppressor, is linked to key events in autophagy and is involved in mitochondrial maintenance² and metabolism.³ DAPK2, which was characterised in 1999,⁴ is significantly smaller than DAPK1, and it lacks ankyrin repeats, the cytoskeletal binding domain and the death domain, all of which are part of DAPK1''s unique structure.¹ Several functions have been ascribed to DAPK2 and they often coincide with those of DAPK1. Like DAPK1, DAPK2 is also involved in the formation of autophagic vesicles,^{5, 6} modulation of receptor induced cell death^{7, 8, 9} and several modes of intrinsic apoptotic cell death.⁶ While epigenetic silencing of DAPK1 has been reported in many different human cancers,^{10, 11} DAPK2 appears to be silenced mainly in haematological disorders,¹² although it has been shown to modulate TRAIL-induced apoptosis in several cancer cell lines of non-haematological origin.⁹ Most approaches used for studying the role of DAPK2 used tagged DAPK2 and it is, therefore, still unclear whether these functions are also carried out by the native protein, expressed at much lower, endogenous, levels.DAPK1 has been shown to regulate mitochondrial integrity and to modulate the mitochondrial membrane potential² but, to the best of our knowledge, no work has been carried out in this respect with regard to DAPK2. Since DAPK1 and DAPK2 appear to share many functions and both are thought to reside, at least partially, in the mitochondria, we hypothesised that DAPK2 depletion regulated mitochondrial metabolism. Mitochondrial dysfunction is characterised by the induction of reactive oxygen species (ROS) in the cell.¹³ Ultimately, dysfunctional mitochondria can no longer be powerhouses of use to the cell and are, therefore, targeted for degradation. Alternatively, their membranes can depolarise leading to the release of cytochrome c, an early apoptotic process.¹⁴ Using two distinct cancer cell types, namely U2OS osteosarcoma and A549 non-small cell lung cancer cells,^{9, 15} we show that DAPK2 depletion increases the levels of intracellular ROS, leads to mitochondrial depolarisation and impairs mitochondrial metabolism. DAPK2 thus exerts metabolic and mitochondria-regulating functions, which have not been described to date and that can explain why it is downregulated in haematological malignancies,^{12, 16, 17} and involved in modulating death-inducing signalling in solid tumours.⁹