ECOTYPIC VARIATION IN CYSTOCLONIUM PURPUREUM (RHODOPHYTA) SYNCHRONIZES LIFE HISTORY EVENTS IN DIFFERENT REGIONS1

Temperature and daylength responses were determined in culture for isolates of the red alga Cystoclonium purpureum (Hudson) Batters from Nova Scotia (NS, Canada), Helgoland (HE, Germany), and Roscoff (RO, France). Most isolates survived temperatures of –1.5°/–2° to 23°C, whereas 25°C was lethal. Only the RO-gametophytes died at 23°C. Optimal growth conditions were 10°–20°C in both long and short days for the NS isolates and 8°–15°C and 8°–18°C at daylengths of >12 h for the RO and HE isolates, respectively. Tetrasporophytes and gametophytes of the NS isolate reproduced at 10°–20°C in long and short days within 5 months. At lower temperatures reproduction was limited or slow. The European isolates formed tetrasporangia at 10°–20°C (HE) or 5°–l8°C(RO), spermatangia at 5°–15°C (HE) or 5°–20°C (RO), and carpospores at 5°–15°C(HE) or 10°–15°C (RO). Short days either blocked or delayed reproduction of the European isolates. The phenology of C. purpureum was studied at Helgoland and Roscoff, where similar seasonal patterns were observed. In early spring, growth was rapid and plants started to form reproductive structures. In summer, tetra-and carpospores were shed followed by degeneration of the upright axes while branched holdfasts persisted. New upright axes and juvenile plants were formed in autumn, but these remained small during the winter months. Published data indicate that the seasonal pattern at Nova Scotia is similar, although the onset of growth and reproduction is delayed until the end of spring. These observations correspond well with the results of the experiments. The life history of C. purpureum is regulated by temperature and daylength. In the eastern Atlantic, the limiting effect of short days confines growth and reproduction to spring and summer. In the western Atlantic, low winter temperatures alone bring about the same seasonal pattern. After plants have reproduced, uprights degenerate in spite of continuing favorable conditions.     

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - DTT dithiothreitol - SDS sodium dodecyl sulphate - TPP thiamin pyrophosphate - DHAS dihydroxyacetone synthase - GSH reduced glutathione     

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.     

Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-alpha spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-alpha value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-alpha concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-alpha levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.     

Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.     

Itinerary of hepatitis B viruses: delineation of restriction points critical for infectious entry

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10(4) per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.     

Inhibition of mitochondrial respiration by the flavone aglycone isovitexin causes aberrant petal and leaf morphology in Silene latifolia

The morphological mutant isovitexin in Silene latifolia (the white campion) has small and up-curled petals and leaves. In this mutant the aglycone isovitexin is the only flavone present in the vacuole. In the present study it is shown that isovitexin has a strong toxic effect on mitochondria that is to a large extent abolished by glycosylation. This effect can be used to explain the aberrant morphology. Isovitexin acts at the level of the ubiquinone pool; cytochrome c — cytochrome aa₃ oxidase activity was unaffected, and with either reduced nicotinamide adenine dinucleotide or succinate as a respiratory substrate, effects on respiration were found in Silene leaves-, potato (Solanum tuberosum) tuber- and sweet potato (Ipomoea batata L.) tuber mitochondria. Since in sweet potato electron transport via the cyanide insensitive pathway was also inhibited, with the ubiquinone pool as the only component (besides the dehydrogenases) shared by these two pathways, the site of inhibition must be at this level.Abbreviations BSA bovine serum albumin - Q ubiquinone - QH₂ reduced ubiquinone - RC respiratory control ratio - SD standard deviation - P/O ratio of molecules ADP phosphorylated/atom oxygen oxidized - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

The Role of the Alternative Oxidase in Stabilizing the in Vivo Reduction State of the Ubiquinone Pool and the Activation State of the Alternative Oxidase

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Q_r/Q_t), thereby avoiding an increase in free radical production. If the Q_r/Q_t were stabilized by the alternative pathway, then Q_r/Q_t should be less stable when the alternative pathway is blocked. Q_r/Q_t increased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Q_r/Q_t increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Q_r/Q_t. Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Q_r/Q_t on the activity of the alternative pathway in intact roots is discussed.