Development of transgenic Caenorhabditis elegans expressing human transthyretin as a model for drug screening.

Familial amyloid polyneuropathy is a hereditary systemic amyloidosis caused by a mutation in the transthyretin (TTR) gene. Amyloid deposits in tissues of patients contain not only full-length TTR but also C-terminal TTR fragments. However, in vivo models to evaluate the pathogenicity of TTR fragments have not yet been developed. Here, we generated transgenic Caenorhabditis elegans strains expressing several types of TTR fragments or full-length TTR fused to enhanced green fluorescent protein in the body wall muscle cells and analyzed the phenotypes of the worms. The transgenic strain expressing residues 81-127 of TTR, which included the ß-strands F and H, formed aggregates and caused defective worm motility and a significantly shortened lifespan compared with other strains. These findings suggest that the C-terminal fragments of TTR may contribute to cytotoxicity of TTR amyloidosis in vivo. By using this C. elegans model system, we found that (-)-epigallocatechin-3-gallate, a major polyphenol in green tea, significantly inhibited the formation of aggregates, the defective motility, and the shortened lifespan caused by residues 81-127 of TTR. These results suggest that our newly developed C. elegans model system will be useful for in vivo pathological analyses of TTR amyloidosis as well as drug screening.

Tamarindus indica extract alters release of alpha enolase, apolipoprotein A-I, transthyretin and Rab GDP dissociation inhibitor beta from HepG2 cells.

BACKGROUND: The plasma cholesterol and triacylglycerol lowering effects of Tamarindus indica extract have been previously described. We have also shown that the methanol extract of T. indica fruit pulp altered the expression of lipid-associated genes including ABCG5 and APOAI in HepG2 cells. In the present study, effects of the same extract on the release of proteins from the cells were investigated using the proteomics approach. METHODOLOGY/PRINCIPAL FINDINGS: When culture media of HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp were subjected to 2-dimensional gel electrophoresis, the expression of seven proteins was found to be significantly different (p<0.03125). Five of the spots were subsequently identified as alpha enolase (ENO1), transthyretin (TTR), apolipoprotein A-I (ApoA-I; two isoforms), and rab GDP dissociation inhibitor beta (GDI-2). A functional network of lipid metabolism, molecular transport and small molecule biochemistry that interconnects the three latter proteins with the interactomes was identified using the Ingenuity Pathways Analysis software. CONCLUSION/SIGNIFICANCE: The methanol extract of T. indica fruit pulp altered the release of ENO1, ApoA-I, TTR and GDI-2 from HepG2 cells. Our results provide support on the effect of T. indica extract on cellular lipid metabolism, particularly that of cholesterol.

Changes in the expression of transthyretin and protein kinase Cγ genes in the prefrontal cortex in response to naltrexone.

Naltrexone, an opioid receptor antagonist, has been approved for clinical use in the treatment of alcohol dependence. In the present study, we examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200µl (16mg/kg) of naltrexone for 4 weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity Pathway Analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and hippocampus. We found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the expression of transthyretin (TTR) and protein kinase C (PKC)γ were increased in the PFC but not in the hippocampus of naltrexone-treated mice. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice. These findings provide a basis for conducting pharmacogenetic research on the effect of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders.

VKORC1 pharmacogenetics and pharmacoproteomics in patients on warfarin anticoagulant therapy: transthyretin precursor as a potential biomarker.

BACKGROUND: Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke. METHODS AND FINDING: We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation. CONCLUSION: This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism.

Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis.

Transthyretin (TTR) tetramer dissociation, misfolding and misassembly are required for the process of amyloid fibril formation associated with familial amyloid polyneuropathy (FAP). Preferential stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Two NSAIDs, diflunisal and flufenamic acid, and trivalent chromium have this ability. Here, we investigated the feasibility of using these molecules for the treatment of FAP utilizing serum samples from 37 FAP patients with 10 different mutations. We demonstrated that the TTR heterotetramer structures in FAP patients serum are significantly less stable than that in normal subjects, indicating the instability of the variant TTR structure is a fundamental cause of TTR amyloidosis. We also demonstrated that therapeutic serum concentrations of diflunisal (100-200 microM) stabilized serum variant TTR tetramer better than those of flufenamic acid (35-70 microM). Trivalent chromium at levels obtained by oral supplementation did not stabilize TTR in a statistically significant fashion. Importantly, diflunisal increased serum TTR stability in FAP patients beyond the level of normal controls.

Hypothalamic gene expression in reproductively photoresponsive and photorefractory Siberian hamsters.

An interval timing mechanism in the brain governs reproduction in seasonally breeding mammals by triggering refractoriness to inhibitory short photoperiods during midwinter. The neural mechanisms responsible for the timing and induction of photorefractoriness by this seasonal clock are unknown. Using cDNA microarrays and RT-PCR, we identified a class of genes encoding thyroxine (T4)-binding proteins (transthyretin, T4-binding globulin, albumin) whose expression is associated with reproductive refractoriness to short day lengths. Down-regulation of these genes was associated with reduced hypothalamic T4 uptake, which was reversed by long-day photoperiod treatments that restored responsiveness to short days. Circulating T4 concentrations did not vary with states of photoresponsiveness in euthyroid hamsters, but blockade of thyroid function accelerated the onset of photorefractoriness to short days. These data link changes in gene expression in the hypothalamus to the functional output of a seasonal clock. Reproductive inhibition in short days depends on T4 only late in the nonbreeding season. Down-regulation of genes encoding T4-binding proteins in the hypothalamus during this interval may restrict access of a static T4 signal to hypothalamic target tissues that regulate reproduction, thereby timing annual transitions in reproductive function. Hypothalamic autoregulation of T4 influx may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism by which neural targets gain access to peripheral hormones.

[Effect of recombinant human growth hormone with total parenteral nutrition on albumin synthesis in patients with peritoneal sepsis].

OBJECTIVE: To investigate the effect of recombinant human growth hormone (rhGH) in combination with total parenteral nutrition (TPN) on albumin synthesis in patients with peritoneal sepsis. METHOD: 17 patients with peritoneal sepsis were divided randomly into two groups. The control group received TPN only for 7 days, and the GH group received both rhGH (12 U/d) and TPN for 7 days. The TPN scheme and other treatment were the same in the two groups. RESULT: Serum albumin, prealbumin, and transferrin concentration were increased in patients in the GH group (P < 0.01), but no apparent effect was observed in the control group (P > 0.05). CONCLUSION: In condition of serious peritoneal sepsis, TPN can not increase albumin, prealbumin, and transferrin synthesis alone, whereas rhGH in combination with TPN significantly increase the synthesis of visceral proteins.

The human lacrimal gland synthesizes apolipoprotein D mRNA in addition to tear prealbumin mRNA, both species encoding members of the lipocalin superfamily.

Apolipoprotein D (apoD), a glycoprotein originally characterized as a component of the high density lipoprotein fraction of human plasma and known to be a member of the lipocalin protein superfamily, has been found in human tear fluid by Western blot analysis. Unlike serum it seems that in the tear fluid apoD exists mainly as a disulphide linked homodimer which is not associated with lecithin/cholesterol acyltransferase (LCAT) or apolipoprotein A-I (apo A-I). By reverse-transcription-PCR (RT-PCR) of mRNA extracted from a human lacrimal gland and use of specific primers we could demonstrate expression of the apoD gene in this tissue. The amplified cDNA was cloned and a subsequent sequence analysis confirmed the identity of apoD mRNA in the human lacrimal gland. These investigations indicate that the lacrimal gland is the site of synthesis of the tear fluid apoD. Although the physiological function of apoD is unknown, it has the ability to bind phospholipids, cholesterol and other small hydrophobic molecules. Therefore, this protein might interact with meibomian lipids present in human tear fluid and probably contribute to the surface spreading of these lipids or it may function as a clearance factor, protecting the cornea from harmful lipophilic molecules.

Transthyretin gene expression in choroid plexus first evolved in reptiles.

The presence of transthyretin in mammals and birds, but not amphibia, suggested that transthyretin expression first appeared in stem reptiles. Therefore, transthyretin synthesis was studied in a lizard. Transthyretin synthesis in choroid plexus pieces from Tiliqua rugosa was demonstrated by incorporation of radiactive amino acids. Oligonucleotides corresponding to conserved regions of transthyretin were used as primers in polymerase chain reaction with lizard choroid plexus cDNA. Amplified DNA was used to screen a lizard choroid plexus cDNA library. A full-length transthyretin cDNA clone was isolated and sequenced. A three-dimensional model of lizard transthyretin was obtained by homology modeling. The central channel of transthyretin, containing the thyroxine-binding site, was found to be completely conserved between reptiles and mammals. Transthyretin expression was not detected in lizard liver. These data suggest that transthyretin first evolved in the choroid plexus of the brain. Due to a change in tissue distribution of gene expression, occurring much later during evolution, transthyretin also became a plasma protein, synthesized in the liver.