Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an α,α-1,1-glycosidic linkage. The best known and most widely distributed pathway of trehalose synthesis involves the transfer of glucose from UDP-glucose to glucose 6-phosphate to form trehalose-6-phosphate and UDP via the trehalose-6-phosphate synthase (TPS1). Trehalose-6-phosphate phosphatase (TPS2) then converts trehalose-6-phosphate to free trehalose. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates and plants, and because of its particular physical features, trehalose is able to protect the integrity of cells against a variety of environmental stresses such as desiccation, dehydration, heat, cold and oxidation. Our current studies described here indicate that trehalose protects Drosophila and mammalian cells from hypoxic and anoxic injury. The mechanism of this protection is probably related to a decrease in protein denaturation through protein-trehalose interactions.

Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the Pelement rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.

Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. Molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that changes in gene expression could help to delineate such mechanisms. The current study used a neonatal mouse model in CCH or CIH combined with cDNA microarrays to determine changes in gene expression in the CCH or CIH mouse heart. Both CCH and CIH induced substantial alterations in gene expression. In addition, a robust right ventricular hypertrophy and cardiac enlargement was found in CCH- but not in CIH-treated mouse heart. On one hand, upregulation in RNA and protein levels of eukaryotic translation initiation factor-2alpha and -4E (eIF-2alpha and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed.

Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(؊)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(؊)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(؊) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O 2 ). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O 2 . The amount of Na ؉ -K ؉ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.The sensitivity of organisms to hypoxia varies, with most mammals being extremely vulnerable to O 2 deprivation. Irreversible injury may occur in mammalian tissues within 5-10 min of severe hypoxia or ischemia, whereas animals such as the turtle, Pseudemys scripta elegans, and invertebrates such as Drosophila melanogaster can recover from hours of experimental O 2 deprivation without a trace of injury. Some of the reasons for tolerance to low O 2 are as follows. (a) Hypoxia induces a decrease in membrane permeability (i.e. ion "channel arrest") that dramatically reduces the energy costs of ion flux (1). (b) These organisms, especially the turtle, have an unusually well developed capacity for rapid entry into, and return from, metabolically depressed steady states using anaerobic metabolism to sustain reduced rates of energy turnover during hypoxia. Clearly, the net effect of this regulated metabolic depression is that it conserves fermentable fuel, reduces deleterious endproduct formation, and extends survival time. (c) The ability to re-allocate cellular energy to...

An acquired mutation in Janus kinase 2 (JAK2), V617F, has recently been identified in human myeloproliferative disorders. Detection of the mutation is helpful in differential diagnosis, prognosis, and predication of therapeutic response. Because the mutation can be present in a small proportion of granulocytic populations in myeloproliferative disorder patients, a highly sensitive detection method is required. In this study, we systematically optimized the reaction conditions of a published amplification refractory mutation system-polymerase chain reaction research protocol to make it a robust clinical diagnostic test. The modifications led to a clear demonstration of the V617F mutation in a patient who would have been easily missed by the original amplification refractory mutation system-polymerase chain reaction assay. The test detects the V617F mutation not only with a high analytic sensitivity of 0.05 to 0.1% but also with a high diagnostic specificity of 99%. In addition, the assay has the ability to distinguish cases with only mutant alleles from cases with mixed normal and mutant alleles. The assay is fast and easy to perform, and no special equipment other than thermocyclers is required. All these features make the assay readily and broadly applicable in clinical molecular diagnostic laboratories. An acquired mutation in the JAK2 gene has recently been described in human myeloproliferative disorders. [1][2][3][4][5] JAK2 is a cytoplasmic tyrosine kinase that plays an essential role in the signaling pathways of cytokines and growth factors. The mutation 1849 GϾT, which leads to amino acid substitution of phenylalanine for a highly conserved valine (V617F), renders JAK2 kinase constitutively active and leads to cell proliferation in the absence of the growth factors. 2,4,5The V617F mutation has been detected in 65 to 97% of patients with polycythemia vera, 23 to 57% of those with essential thrombocythemia (ET), and 30 to 57% of idiopathic myelofibrosis.