In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.

Trehalose is potentially a useful cryo-or anhydroprotectant molecule for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. In this study, we isolated and characterized the functions of a facilitated trehalose transporter [trehalose transporter 1 (TRET1)] from an anhydrobiotic insect, Polypedilum vanderplanki. Tret1 cDNA encodes a 504-aa protein with 12 predicted transmembrane structures. Tret1 expression was induced by either desiccation or salinity stress. Expression was predominant in the fat body and occurred concomitantly with the accumulation of trehalose, indicating that TRET1 is involved in transporting trehalose synthesized in the fat body into the hemolymph. Functional expression of TRET1 in Xenopus oocytes showed that transport activity was stereochemically specific for trehalose and independent of extracellular pH (between 4.0 and 9.0) and electrochemical membrane potential. These results indicate that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. The extraordinarily high values for apparent K m (>100 mM) and Vmax (>500 pmol/min per oocyte) for trehalose both indicate that TRET1 is a high-capacity transporter of trehalose. Furthermore, TRET1 was found to function in mammalian cells, suggesting that it confers trehalose permeability on cells, including those of vertebrates as well as insects. These characteristic features imply that TRET1 in combination with trehalose has high potential for basic and practical applications in vivo.anhydrobiosis ͉ insect ͉ trehalose transport ͉ Polypedilum vanderplanki dessication-inducible gene

Anhydrobiosis is an extremely dehydrated state in which organisms show no detectable metabolism but retain the ability to revive after rehydration. Thus far, two hypotheses have been proposed to explain how cells are protected during dehydration: (i) water replacement by compatible solutes and (ii) vitrification. The present study provides direct physiological and physicochemical evidence for these hypotheses in an African chironomid, Polypedilum vanderplanki, which is the largest multicellular animal capable of anhydrobiosis. Differential scanning calorimetry measurements and Fourier-transform infrared (FTIR) analyses indicated that the anhydrobiotic larvae were in a glassy state up to as high as 65°C. Changing from the glassy to the rubbery state by either heating or allowing slight moisture uptake greatly decreased the survival rate of dehydrated larvae. In addition, FTIR spectra showed that sugars formed hydrogen bonds with phospholipids and that membranes remained in the liquid-crystalline state in the anhydrobiotic larvae. These results indicate that larvae of P. vanderplanki survive extreme dehydration by replacing the normal intracellular medium with a biological glass. When entering anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body by FTIR microscopic mapping image. Therefore, we assume that trehalose plays important roles in water replacement and intracellular glass formation, although other compounds are surely involved in these phenomena.trehalose ͉ water replacement ͉ Fourier-transform infrared microspectroscopy ͉ biological glass ͉ cryptobiosis

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O2•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.