Cell cycle arrest and apoptosis induction by methanolic leaves extracts of four Annonaceae plants.

BACKGROUND: Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders and Dasymaschalon sp. have been used for traditional medicine to treat cancer-like symptoms in some ethnic groups of Thailand and Laos. METHODS: We evaluated the anti-cancer activity of these Annonaceae plants against several human cancer cell lines. The apoptosis induction was detected by Annexin/propidium iodide (PI) staining. Phytochemical screening was tested by standard protocols and bioactive compounds were determined by HPLC. RESULTS: The crude extracts from leaves of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum showed particular effects that were found to vary depending on the cancer cell line, suggesting that the effect was in a cell-type specific manner. Interestingly, the induction of apoptotic cell death was prominent by the leaves-derived crude extract of M. modestum. This crude was, therefore, subjected to cell cycle analysis by PI staining. Results showed that this crude extract arrested cell cycle and increased the percentage of cells in the SubG1 phase in some cancer cell lines. The phytochemical screening tests indicated that all crude extracts contained tannins and flavonoids. HPLC of flavonoids using standards identified rutin as an active compound in U. longipes and Dasymaschalon sp., whereas quercetin was found in U. longipes and M. modestum. CONCLUSIONS: These crude extracts provide a new source for rutin and quercetin, which might be capable of inducing cancer cell apoptotic death in a cell-type specific manner. This suggests, by analyzing the major bioactive compounds, the potential use of these crudes for chemotherapy in the future.

Dysregulation of ferroportin gene expression in ß(0)-thalassemia/Hb E disease.

During erythropoiesis, iron levels need to be carefully regulated to ensure there is sufficient iron available for hemoglobin synthesis, but that there is no excess to cause damage to the developing erythroblast. Iron influx to the developing erythroblast is controlled by the expression of the transferrin receptor, while iron efflux is regulated by ferroportin (FPN), the sole iron-exporting protein. FPN is encoded through multiple messenger RNAs (mRNAs) some of which contain an iron-responsive element (variant I mRNAs) and some of which do not (variant II mRNAs). This study sought to investigate the expression of the FPN mRNAs in developing erythroblasts from normal controls and ß(0)-thalassemia/Hb E patients. While levels of FPN protein were relatively constant, marked reductions of the variant I message were seen in erythroblasts from ß(0)-thalassemia/Hb E patients as compared to normal control cells, particularly in late erythropoiesis. Variant II mRNAs were generally increased during erythroid differentiation. No difference was seen in levels of either transferrin or ferritin heavy chain expression. While no difference was observed in labile iron pools under normal culture conditions, erythroblasts from ß(0)-thalassemia/Hb E patients showed a significantly reduced expression of total FPN message under high iron conditions as compared to normal control erythroblasts. These results are consisted with dysregulation of iron efflux from the maturing erythroblast in ß(0)-thalassemia/Hb E patients, and this dysregulation possibly contributes to ineffective erythropoiesis seen in these patients.

A 2D-proteomic analysis identifies proteins differentially regulated by two different dengue virus serotypes.

The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

In-vitro antimalarial activity of methanolic leaf- and stem-derived extracts from four Annonaceae plants.

OBJECTIVE: Plants in the Annonaceae family are known for having abundant biologically active secondary metabolites. They have been used in alternative drugs for various diseases in several countries, for instance, the bark of Cananga odorata (Lam.) Hook and Thomson is used for Ophthalmic inflammation and wound healing in Malaysia. Extracts from the leaves and stems of four Annonaceae plants, namely Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Dasymaschalon sp., Artabotrys burmanicus A.DC, and Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders were investigated for growth inhibitory activity against blood-stage Plasmodium falciparum growth in vitro and for non-specific cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). Antimalarial activity was assessed by invasion inhibition assay and the percentage of infected red blood cells on blood smears were determined. Cytotoxicity was tested by culturing PBMCs with the extracts, and viabilities were determined by Annexin V/propidium iodide staining. RESULTS: A. burmanicus stem extract and M. modestum leaf extract were capable of inhibiting growth of P. falciparum when used at 200 µg/mL compared to chloroquine. The extracts at effective concentrations, did not affect the viability of PBMCs. These results support further need for characterization of active compounds from specific Annonaceae plants in order to exploit their components for potential malaria treatment.

The anti-leukemic activity of a luteolin-apigenin enriched fraction from an edible and ethnomedicinal plant, Elsholtzia stachyodes, is exerted through an ER stress/autophagy/cell cycle arrest/ apoptotic cell death signaling axis.

BACKGROUND: Elsholtzia is a genus in the family Lamiaceae, and some species in this genus are commonly used for food and in ethnomedicinal formulations by some ethnic groups of China and Thailand. Despite their apparent utility, few studies have been conducted to evaluate their potential as sources of medicinally active agents. PURPOSE: We aimed to investigate the cytotoxicity of ethanolic extracts from three selected edible plant species of the genus Elsholtzia and the most promising extract was further characterized for the bioactive constituents and signaling mechanisms associated with the anti-leukemic activity. MATERIALS AND METHODS: Ethanolic extracts were screened for cytotoxicity using flow cytometry. HPLC and LC-MS were used to analyze the chemical constituents of the most potent fraction from E. stachyodes. The relevant mechanism of action was assessed by western blot and multispectral imaging flow cytometry (MIFC). RESULTS: The most potent anti-leukemic activity was observed with the ethanolic extract from E. stachyodes. Luteolin and apigenin were characterized as the major constituents in the fraction from E. stachyodes. Mechanistically, the luteolin-apigenin enriched fraction (LAEF) induced the UPR, increased autophagic flux, induced cell cycle arrest and apoptotic cell death. LAEF showed significantly less cytotoxicity towards peripheral blood mononuclear cells (PBMCs) as compared to leukemia cell lines. CONCLUSION: This study is the first to report E. stachyodes as a new source of luteolin and apigenin which are capable of triggering leukemic cell death. This could lead to a novel strategy against leukemia using ethnomedicinal plant extracts as an alternative or supplemental anti-cancer agent.

Proteomic analysis of hemoglobin H-constant spring (Hb H-CS) erythroblasts.

Hemoglobin H disease (Hb H) arises through the loss or dysfunction of three of the four alpha globin genes through the co-inheritance of either gross gene deletions or an abnormal hemoglobin which causes a non-deletional loss of α-globin expression. This study sought to investigate erythropoiesis in Hb H-Constant Spring (Hb H-CS) disease, a common form of Hb H disease in Southeast Asia, caused by the inheritance of the Constant Spring variant hemoglobin together with deletion of two of the alpha globin genes. In comparison to normal erythroblasts, Hb H-CS erythroblasts showed reduced cell expansion although no difference in differentiation was observed. Proteomic analysis revealed the increased expression of both chaperone and chaperonin proteins as well as down regulation of proteins regulating apoptosis. Both chaperone and chaperonin mediated folding require ATP, and evidence of increase energy demand was seen in the form of increased expression of enzymes involved in purine biosynthesis and increased levels of reactive oxygen species. A significant increase in apoptosis was seen in Hb H-CS erythroblasts, and the results from the proteomic analysis suggest that this arises at least in part from the consequences of increased folding requirements in the Hb H-CS erythroblast.

An IgM monoclonal antibody against domain 1 of CD147 induces non-canonical RIPK-independent necroptosis in a cell type specific manner in hepatocellular carcinoma cells.

CD147/Basigin/EMMPRIN is overexpressed in several cancerous tissues and it has been shown to induce matrix metalloproteinases (MMPs) whose expression is associated with cancer metastasis. Thus, targeting CD147 with monoclonal antibodies (mAbs) potentially has therapeutic applications in cancer immunotherapy. Here, we report the use of anti-CD147 mAbs targeting domain 1 of CD147, namely M6-1D4 (IgM), M6-1F3 (IgM), M6-2F9 (IgM) and M6-1E9 (IgG2a), against several human cancer cell lines. Strikingly, IgM but not IgG mAbs against CD147, especially clone M6-1D4, induced acute cellular swelling, and this phenomenon appeared to be specifically found with hepatocellular carcinoma (HCC) cells. Furthermore, molecular investigation upon treating HepG2 cells with M6-1D4 showed unfolded protein response (UPR) activation, autophagosome accumulation, and cell cycle arrest, but without classic apoptosis related features. More interestingly, prolonged M6-1D4 treatment (24 h) resulted in irreversible oncosis leading to necroptosis. Furthermore, treatment with a mixed lineage kinase domain-like psuedokinase (MLKL) inhibitor and partial knockout of MLKL resulted in reduced sensitivity to necroptosis in M6-1D4-treated HepG2 cells. Surprisingly however, the observed necroptotic signaling axis appeared to be non-canonical as it was independent of receptor-interacting serine/threonine-protein kinase (RIPK) phosphorylation. In addition, no cytotoxic effect on human dermal fibroblast (HDF) was observed after incubation with M6-1D4. Taken together, this study provides clues to target CD147 in HCC using mAbs, as well as sheds new light on a novel strategy to kill cancerous cells by the induction of necroptosis.

Increased oxidative metabolism is associated with erythroid precursor expansion in ß0-thalassaemia/Hb E disease.

Erythropoiesis in ß0-thalassaemia/Hb E patients, the most common variant form of ß-thalassaemia in Southeast Asia, is characterized by accelerated differentiation and over-expansion of erythroid precursor cells. The mechanism driving this accelerated expansion and differentiation remain unknown. To address this issue a proteomic analysis was undertaken to firstly identify proteins differentially expressed during erythroblast differentiation and a second analysis was undertaken to identify proteins differentially expressed between ß0-thalassaemia/Hb E erythroblasts and control erythroblasts. The majority of proteins identified as being differentially expressed between ß0-thalassaemia/Hb E and control erythroblasts were constituents of the glycolysis/TCA pathway and levels of oxidative stress correlated with the degree of erythroid expansion. A model was constructed linking these observations with previous studies showing increased phosphorylation of ERK1/2 in thalassemic erythroblasts which predicted the increased activation of PKA, PKB and PKC which Western analysis confirmed. Inhibition of PKA or PKC reduced ß0-thalassaemia/Hb E erythroblast differentiation and/or expansion. We propose that increased expansion and differentiation of ß0-thalassaemia/Hb E erythroblasts occur as a result of feedback loops acting through increased oxidative metabolism.

Enhanced activation of autophagy in ß-thalassemia/Hb E erythroblasts during erythropoiesis.

Erythropoiesis in ß-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and ß-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with L: -asparagine. A significantly higher level of autophagy was seen in ß-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca(2+) as modulation of Ca(2+) levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca(2+) levels. These results suggest that high levels of autophagy in ß-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in ß-thalassemia/Hb E erythroblasts.

A mechanism of ineffective erythropoiesis in ß-thalassemia/Hb E disease.

BACKGROUND: Cells respond to stress stimuli through a number of response pathways, of which one of the most important and well characterized is the unfolded protein response. Despite a large body of work which suggests that stress in erythroblasts may play a pivotal role in the pathogenesis of beta-thalassemia/Hb E disease, this pathway remains uninvestigated. DESIGN AND METHODS: Day 10 erythroblasts from normal controls and beta-thalassemia/Hb E patients were subjected to internal (treatment with tunicamycin) and external (serum and growth factor withdrawal) stress stimuli and the activation of the unfolded protein response pathway was investigated. RESULTS: Normal erythroblasts responded to both internal and external stress by activating the unfolded protein response (UPR) pathway while in contrast, erythroblasts from beta-thalassemia/Hb E patients only showed activation of the unfolded protein response pathway in response to internal stress. This was reflected by a markedly increased induction of apoptosis in serum and growth factor deprived beta-thalassemia/Hb E erythroblasts as compared to control cells. Modulation of the levels of intracellular Ca(2+) in thalassemic erythroblasts restored UPR activation during serum deprivation and significantly reduced the level of serum deprivation induced apoptosis to control levels. CONCLUSIONS: These results suggest the failure of thalassemic erythroblasts to cope with cellular stress caused by an impaired UPR function as a result of high Ca(2+) levels may exacerbate thalassemic cell death during erythropoiesis.

Increased erythropoiesis of beta-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation.

Beta-thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of beta-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34+ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca2+ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in beta-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca2+. Modulation of either cAMP or Ca2+ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of beta-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca2+.

Cytotoxic and cytostatic effects of four Annonaceae plants on human cancer cell lines.

Several species of the Annonaceae plants have been used as complementary medicine for cancer-associated illnesses in some ethnic groups of northern Thailand. This study investigated the cytotoxic and cytostatic activity of methanolic extracts derived from the stems of these plants, including Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders, and Dasymaschalon sp. Cell death induction of seven human cancer cell lines and cell cycle analyses were assessed by Annexin V and/or propidium iodide (PI) staining and analyzed by flow cytometry. Treatment of cancer cell lines with the extract of four Annonaceae plants resulted in various cytotoxic activities depending on cell type. The extract of U. longipes exhibited the highest cytotoxic activity capable of inducing cell death of several cancer cell lines, particularly against hepatocellular carcinoma cell lines (HepG2 and Hep3B). This extract was capable of inducing cell cycle arrest at the SubG1 phase. Phytochemical screening of all the extracts revealed the presence of alkaloids, sterols, tannins, anthraquinone glycoside, coumarin, and flavonoids. Determination of active compounds by high-performance liquid chromatography standards revealed bullatacin and asiminecin in all the extracts. The extract of Annonaceae stem or its compounds may provide an opportunity for the development of new therapies against cancer.

Functional characterization of lysosomal interaction of Akt with VRK2.

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy.

A First Phylogeny of the Genus Dimocarpus and Suggestions for Revision of Some Taxa Based on Molecular and Morphological Evidence.

Dimocarpus longan, commonly known as the longan, belongs to the family Sapindaceae, and is one of the most economically important fruits commonly cultivated in several regions in Asia. There are various cultivars of longan throughout the Thai-Malay peninsula region, but until now no phylogenetic analysis has been undertaken to determine the genetic relatedness of these cultivars. To address this issue, 6 loci, namely ITS2, matK, rbcL, trnH-psbA, trnL-I and trnL-trnF were amplified and sequenced from 40 individuals consisting of 26 longan cultivars 2 types of lychee and 8 herbarium samples. The sequencing results were used to construct a phylogenetic tree using the neighbor-joining (NJ), maximum likelihood (ML) and Bayesian inference (BI) criteria. The tree showed cryptic groups of D. longan from the Thailand-Malaysia region (Dimocarpus longan spp.). This is the first report of the genetic relationship of Dimocarpus based on multi-locus molecular markers and morphological characteristics. Multiple sequence alignments, phylogenetic trees and species delimitation support that Dimocarpus longan spp. longan var. obtusus and Dimocarpus longan spp. malesianus var. malesianus should be placed into a higher order and are two additional species in the genus Dimocarpus. Therefore these two species require nomenclatural changes as Dimocarpus malesianus and Dimocarpus obtusus, respectively.

Endogamous marriage and the prevalence of hemoglobin E in ethnic groups of northern Thailand.

OBJECTIVE: To investigate the impact of the endogamous marriage culture on the prevalence of the hemoglobin E (HbE) recessive variant. METHODS: The prevalence of the hemoglobin E (HbE) recessive variant was determined by dot-blot hybridization in 4 endogamous villages (1 Mlabri and 3 Htin ethnic groups) in comparison with 9 other nearby non-endogamous populations. RESULTS: Although the overall HbE prevalence in the population studied (8.44%, 33/391) was not significantly different from that of the general southeast Asian population, a high prevalence and individuals with homozygous HbE were observed in two villages, the Mlabri from Wiang Sa district and the Htin from Thung Chang district of Nan province (26.3% and 26.9%, respectively). The low HbE allelic frequency noticed in some endogamous populations suggests that not only endogamy but also other evolutionary forces, such as founder effect and HbE/ß-thalassemia negative selection may have an effect on the distribution of the HbE trait. CONCLUSION: Our study strongly documents that cultural impact has to be considered in the extensive prevalence studies for genetic disorders in the ethnic groups of northern Thailand.

A comprehensive ethnic-based analysis of alpha thalassaemia allelle frequency in northern Thailand.

Alpha (α)-thalassaemia is one of the most prevalent hereditary blood disorders, commonly affecting Southeast Asian people, with the highest incidence (30-40%) being seen in northern Thailand. However, this high incidence was estimated without consideration of the variations between ethnic populations and the geographical location of the populations. To address this issue, a total of 688 samples from 13 different northern Thai ethnic groups (30 villages) categorized into three linguistic groups were genotyped for deletional alpha-thalassaemia (-α3.7, -α4.2, --SEA and --THAI) and/or non-deletional alpha-thalassaemia (αCS and αPS) via multiplex gap-PCR and dot-blot hybridization, respectively. Alpha+(-α3.7, -α4.2, αCS and αPS) and alpha°-thalassaemia (--SEA and --THAI) allele frequencies (with 95% Confidence Interval) were the highest in the Sino-Tibetan group [0.13 (0.08-0.18)] and the Tai-Kadai group [0.03 (0.02-0.05)], respectively. With regards to ethnicity, the varying allele frequency of α+ and α°-thalassaemia amongst a variety of ethnic groups was observed. The highest α+-thalassaemia allele frequency was found in the Paluang [0.21 (0.10-0.37)] while α°-thalassaemia allele frequency was the highest in the Yuan [0.04 (0.01-0.10)]. These detailed results of alpha thalassaemia allele frequency and genetic diversity amongst the northern Thai ethnic groups demonstrate the need for ethnicity based thalassaemia prevention programs.

Hypermethylation of 28S ribosomal RNA in ß-thalassemia trait carriers.

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of ß-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in ß-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in ß-thalassemia trait carriers.

Analysis of protein profiling studies of ß-thalassemia/Hb E disease.

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in ß-thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha-1-antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium-binding protein, microtubule-actin cross-linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin-2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified "response to inorganic substances" as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of ß-thalassemia/Hb E disease is starting to emerge.

Iron dysregulation in beta-thalassemia.

Iron deficiency anemia and iron overload conditions affect more than one billion people worldwide. Iron homeostasis involves the regulation of cells that export iron into the plasma and cells that utilize or store iron. The cellular iron balance in humans is primarily mediated by the hepcidin-ferroportin axis. Ferroportin is the sole cellular iron export protein, and its expression is regulated transcriptionally, post-transcriptionally and post-translationally. Hepcidin, a hormone produced by liver cells, post-translationally regulates ferroportin expression on iron exporting cells by binding with ferroportin and promoting its internalization by endocytosis and subsequent degradation by lysosomes. Dysregulation of iron homeostasis leading to iron deposition in vital organs is the main cause of death in beta-thalassemia patients. Beta-thalassemia patients show marked hepcidin suppression, ineffective erythropoiesis, anemia and iron overload. Beta-thalassemia is common in the Mediterranean region, Southeast Asia and the Indian subcontinent, and the focus of this review is to provide an update on the factors mediating hepcidin related iron dysregulation in beta-thalassemia disease. Understanding this process may pave the way for new treatments to ameliorate iron overloading and improve the long term prognosis of these patients.