Progressive island colonization and ancient origin of Hawaiian Metrosideros (Myrtaceae)

Knowledge of the evolutionary history of plants that are ecologically dominant in modern ecosystems is critical to understanding the historical development of those ecosystems. Metrosideros is a plant genus found in many ecological and altitudinal zones throughout the Pacific. In the Hawaiian Islands, Metrosideros polymorpha is an ecologically dominant species and is also highly polymorphic in both growth form and ecology. Using 10 non-coding chloroplast regions, we investigated haplotype diversity in the five currently recognized Hawaiian Metrosideros species and an established out-group, Metrosideros collina, from French Polynesia. Multiple haplotype groups were found, but these did not match morphological delimitations. Alternative morphologies sharing the same haplotype, as well as similar morphologies occurring within several distinct island clades, could be the result of developmental plasticity, parallel evolution or chloroplast capture. The geographical structure of the data is consistent with a pattern of age progressive island colonizations and suggests de novo intra-island diversification. If single colonization events resulted in a similar array of morphologies on each island, this would represent parallel radiations within a single, highly polymorphic species. However, we were unable to resolve whether the pattern is instead explained by ancient introgression and incomplete lineage sorting resulting in repeated chloroplast capture. Using several calibration methods, we estimate the colonization of the Hawaiian Islands to be potentially as old as 3.9 (-6.3) Myr with an ancestral position for Kaua'i in the colonization and evolution of Metrosideros in the Hawaiian Islands. This would represent a more ancient arrival of Metrosideros to this region than previous studies have suggested.     

A Prospective Study of Transsulfuration Biomarkers in Autistic Disorders

The goal of this study was to evaluate transsulfuration metabolites in participants diagnosed with autism spectrum disorders (ASDs). Transsulfuration metabolites, including: plasma reduced glutathione (GSH), plasma oxidized glutathione (GSSG), plasma cysteine, plasma taurine, plasma sulfate, and plasma free sulfate among participants diagnosed with ASDs (n = 38) in comparison to age-matched neurotypical controls were prospectively evaluated. Testing was conducted using Vitamin Diagnostics, Inc. (CLIA-approved). Participants diagnosed with ASDs had significantly (P < 0.001) decreased plasma reduced GSH, plasma cysteine, plasma taurine, plasma sulfate, and plasma free sulfate relative to controls. By contrast, participants diagnosed with ASDs had significantly (P < 0.001) increased plasma GSSG relative to controls. The present observations are compatible with increased oxidative stress and a decreased detoxification capacity, particularly of mercury, in patients diagnosed with ASDs. Patients diagnosed with ASDs should be routinely tested to evaluate transsulfuration metabolites, and potential treatment protocols should be evaluated to potentially correct the transsulfuration abnormalities observed. An erratum to this article can be found at     

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

The Niemann‐Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein‐derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl‐CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element‐binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low‐density lipoprotein (LDL)‐derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein‐deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL‐derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL‐derived cholesterol‐dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL‐derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL‐derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis. J. Cell. Biochem. 108: 1102–1116, 2009. © 2009 Wiley‐Liss, Inc.     

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

Effects of long-term exposure to 900 megahertz electromagnetic field on heart morphology and biochemistry of male adolescent rats

The pathological effects of exposure to an electromagnetic field (EMF) during adolescence may be greater than those in adulthood. We investigated the effects of exposure to 900 MHz EMF during adolescence on male adult rats. Twenty-four 21-day-old male rats were divided into three equal groups: control (Cont-Gr), sham (Shm-Gr) and EMF-exposed (EMF-Gr). EMF-Gr rats were placed in an EMF exposure cage (Plexiglas cage) for 1 h/day between postnatal days 21 and 59 and exposed to 900 MHz EMF. Shm-Gr rats were placed inside the Plexiglas cage under the same conditions and for the same duration, but were not exposed to EMF. All animals were sacrificed on postnatal day 60 and the hearts were extracted for microscopic and biochemical analyses. Biochemical analysis showed increased levels of malondialdehyde and superoxide dismutase, and reduced glutathione and catalase levels in EMF-Gr compared to Cont-Gr animals. Hematoxylin and eosin stained sections from EMF-Gr animals exhibited structural changes and capillary congestion in the myocardium. The percentage of apoptotic myocardial cells in EMF-Gr was higher than in either Shm-Gr or Cont-Gr animals. Transmission electron microscopy of myocardial cells of EMF-Gr animals showed altered structure of Z bands, decreased myofilaments and pronounced vacuolization. We found that exposure of male rats to 900 MHz EMF for 1 h/day during adolescence caused oxidative stress, which caused structural alteration of male adolescent rat heart tissue.     

Blood-brain barrier to 6-hydroxydopamine: uptake by heart and brain.

The presumed blood-brain barrier to 6-OH-DA was studied in 25–36 day old rats with high (30 mg/kg) and low (0.14 mg/kg) dose intravenous 6-OH-DA-1-¹⁴C. From measurements of radioactivity in heart, brain and plasma, the authors conclude that a blood-brain barrier to 6-OH-DA exists in this species.     

Pilot Plant Glycerol Production with a Slow-Feed Osmophilic Yeast Fermentation

A slow feed batch fermentation is described for the production of glycerol from sugar. The conversion efficiency was approximately 1 mole of glycerol produced per mole of glucose utilized after the cell growth phase. The glycerol production phase was extended several-fold by periodic glucose addition. The yeast cell count remained constant during this time as limited by phosphate, a deficiency required for an efficient glycerol fermentation. A small amount of phosphate was supplied during the extended fermentation, maintaining an active culture, by the normal autolysis of spent cells. Interfering or inhibitory by-products did not accumulate, and the osmophilic yeasts are tolerant of high glycerol concentrations. These factors combined to allow a particularly efficient fermentation well suited to product enrichment by supplying large quantities of substrate over an extended period.