Inhibition of Intestinal Thiamin Transport in Rat Model of Sepsis.

ObjectivesThiamin deficiency is highly prevalent in patients with sepsis, but the mechanism by which sepsis induces thiamin deficiency is unknown. This study aimed to determine the influence of various severity of sepsis on carrier-mediated intestinal thiamin uptake, level of expressions of thiamin transporters (thiamin transporter-1 and thiamin transporter-2), and mitochondrial thiamin pyrophosphate transporter.DesignRandomized controlled study.SettingResearch laboratory at a Veterans Affairs Medical Center.SubjectsTwenty-four Sprague-Dawley rats were randomized into controls, mild, moderate, and severe sepsis with equal number of animals in each group.InterventionsSepsis was induced by cecal ligation and puncture with the cecum ligated below the cecal valve at 25%, 50%, and 75% of cecal length, defined as severe, moderate, and mild sepsis, respectively. Control animals underwent laparotomy only.Measurements and main resultsAfter 2 days of induced sepsis, carrier-mediated intestinal thiamin uptake was measured using [H]thiamin. Expressions of thiamin transporter-1, thiamin transporter-2, and mitochondrial thiamin pyrophosphate transporter proteins and messenger RNA were measured. Proinflammatory cytokines (interleukin-1β and interleukin-6) and adenosine triphosphate were also measured. Sepsis inhibited [H]thiamin uptake, and the inhibition was a function of sepsis severity. Both cell membrane thiamin transporters and mitochondrial thiamin pyrophosphate transporter expression levels were suppressed; also levels of adenosine triphosphate in the intestine of animals with moderate and severe sepsis were significantly lower than that of sham-operated controls.ConclusionsFor the first time, we demonstrated that sepsis inhibited carrier-mediated intestinal thiamin uptake as a function of sepsis severity, suppressed thiamin transporters and mitochondrial thiamin pyrophosphate transporter, leading to adenosine triphosphate depletion

Identification of transporter genes from the fungal endophyte Neotyphodium lolii : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Plant Biology at Massey University, Palmerston North, New Zealand /

Neotyphodium lolii is an endophytic fungus that lives in the pasture grass, Lolium perenne. They share a mutualistic symbiotic relationship. N. lolii lives out its life cycle within the plant and produces secondary metabolites, including alkaloids peramine, ergovaline and lolitrem which protect the grass from insect and animal herbivory. In fungi the biosynthetic genes of secondary metabolites are often located in gene clusters. These clusters frequently contain one or more genes that code for transporter proteins responsible for the removal of toxic products from the fungal cells. Plants produce defence compounds, including antifungals to protect themselves from colonising fungi. However endophytes are able to neutralise these host toxins, one mechanism for this is possibly by efflux through transporter channels. The goal of this study was to identify ABC and MFS genes from N. lolii. These two families are the largest and most diverse of transporter families, which transport a variety of substrates, including peptides, toxins, ions and sugars across membranes. Using degenerate PCR primers designed from fungal multi-drug transporter sequences, four unique ABC gene fragments were amplified from N. lolii. A further two ABC sequences and two MFS gene fragments were identified in a database of N. lolii EST sequences. RT-PCR was used to compare expression of isolated ABC and MFS genes in N. lolii, growing in culture and in infected plants. Up-regulation of transporter transcripts in planta could suggest a role in symbiosis. Some genes were seen to have a visibly different expression pattern from others, although all genes were strongly expressed in cultured mycelia. Gene expression in the plant host was most evident in tissues more heavily infected with endophyte. To discover possible roles for the isolated transporter genes in transporting endophyte secondary metabolites a strain distribution study was completed. Five of the putative ABC and MFS genes were compared against 12 Epichloë and Neotyphodium endophytes. Amplified PCR products in the genotypes screened produced a unique pattern of gene occurrence for each of the five transporters. This added to the characterisation of the transporter genes and showed that one gene, gABC 4e, was the most diverse in its distribution, while another ABC gene gABC 4g was present across all genotypes. One ABC gene (gABC 4e) plus flanking DNA was sequenced in full. Bioinformatic analyses suggested that gABC 4c may be a half sized ABC transporter gene of 2 kb with four exons. An orotate phosphoribosyltransferase was identified 2 kb upstream of the ABC transporter. Further work will be needed to confirm that the start and stop codons of this ABC transporter have been accurately predicted, as well as to verify the putative intron/ exon boundaries identified by gene prediction programmes. The role of N. lolii ABC transporter gABC 4c has not been determined, however future research could focus on the nature of the substrate(s) transported, the sub-cellular location of the channel, and the effects of gene knockout or over-expression on the symbiosis between N. lolii and perennial ryegrass

Support varieties for transporter category algebras

Let G be a finite group. Over any finite G-poset P we may define a transporter category as the corresponding Grothendieck construction. The classifying space of the transporter category is the Borel construction on the G-space BP, while the k-category algebra of the transporter category is the (Gorenstein) skew group algebra on the G-incidence algebra kP. We introduce a support variety theory for the category algebras of transporter categories. It extends Carlson's support variety theory on group cohomology rings to equivariant cohomology rings. In the mean time it provides a class of (usually non selfinjective) algebras to which Snashall-Solberg's (Hochschild) support variety theory applies. Various properties will be developed. Particularly we establish a Quillen stratification for modules.Comment: 22 pages. Removed some small errors. Added a Lemma 2.3.2 and 2 new references on Gorenstein skew group algebra

Roles for the Uptake\u3csub\u3e2\u3c/sub\u3e Transporter OCT3 in Regulation of Dopaminergic Neurotransmission and Behavior

Transporter-mediated uptake determines the peak concentration, duration, and physical spread of released monoamines. Most studies of monoamine clearance focus on the presynaptic uptake1 transporters SERT, NET and DAT. However, recent studies have demonstrated the expression of the uptake2 transporter OCT3 (organic cation transporter 3), throughout the rodent brain. In contrast to NET, DAT and SERT, OCT3 has higher capacity and lower affinity for substrates, is sodium-independent, and is multi-specific, with the capacity to transport norepinephrine, dopamine, serotonin and histamine. OCT3is insensitive to inhibition by cocaine and antidepressant drugs but is inhibited directly by the glucocorticoid hormone corticosterone. Thus, OCT3 represents a novel, stress hormone-sensitive, monoamine transport mechanism. Incorporating this transporter into current models of monoaminergic neurotransmission requires information on: A) the cellular and subcellular localization of the transporter; B) the effects of OCT3 inhibitors on monoamine clearance; and C) the consequences of decreased OCT3-mediated transport on physiology and/or behavior. This review summarizes studies describing the anatomical distribution of OCT3, its cellular and subcellular localization, its contribution to the regulation of dopaminergicsignaling, and its roles in the regulation of behavior. Together, these and other studies suggest that both Uptake1 and Uptake2 transporters play key roles in regulating monoaminergic neurotransmission and the effects of monoamines on behavior

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs

Organic Cation Transporter 3 (OCT3) Is Localized to Intracellular and Surface Membranes in Select Glial and Neuronal Cells Within the Basolateral Amygdaloid Complex of Both Rats and Mice

Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreactivity on axonal, dendritic, and astrocytic processes adjacent to a variety of synapses, as well as on neuronal somata. In addition to plasma membrane sites, OCT3 immunolabeling was also observed associated with neuronal and glial endomembranes, including Golgi, mitochondrial and nuclear membranes. Particularly prominent labeling of the outer nuclear membrane was observed in neuronal, astrocytic, microglial and endothelial perikarya. The localization of OCT3 to neuronal and glial plasma membranes adjacent to synaptic sites is consistent with an important role for this transporter in regulating the amplitude, duration, and physical spread of released monoamines, while its localization to mitochondrial and outer nuclear membranes suggests previously undescribed roles for the transporter in the intracellular disposition of monoamines

An Arabidopsis flavonoid transporter is required for anther dehiscence and pollen development

FLOWER FLAVONOID TRANSPORTER (FFT) encodes a multidrug and toxin efflux family transporter in Arabidopsis thaliana. FFT (AtDTX35) is highly transcribed in floral tissues, the transcript being localized to epidermal guard cells, including those of the anthers, stigma, siliques and nectaries. Mutant analysis demonstrates that the absence of FFT transcript affects flavonoid levels in the plant and that the altered flavonoid metabolism has wide-ranging consequences. Root growth, seed development and germination, and pollen development, release and viability are all affected. Spectrometry of mutant versus wild-type flowers shows altered levels of a glycosylated flavonol whereas anthocyanin seems unlikely to be the substrate as previously speculated. Thus, as well as adding FFT to the incompletely described flavonoid transport network, it is found that correct reproductive development in Arabidopsis is perturbed when this particular transporter is missing

Identifying the transporters of different flavonoids in plants

We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT’s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologues within the large Arabidopsis MATE family