Genetic variations of CD36 and low platelet CD36 expression - a risk factor for lipemic plasma donation in Taiwanese apheresis donors.

BACKGROUND: New CD36 mutations are constantly being identified, although no study has specifically targeted a Taiwanese population. CD36 deficiency can result in dyslipid state and slow clearance of chylomicron. This could be linked to more frequent lipemic donations. STUDY DESIGN AND METHODS: We used flow cytometric methods to study the CD36 deficiency in 640 regular volunteer platelet apheresis donors from Taipei blood centre. The coding exons of CD36 gene were sequenced in CD36-deficient individuals, and the allele frequencies of CD36 variants were determined in the larger population by mutation-specific PCR and oligonucleotide hybridization. Visual inspection of lipemic plasma was routinely performed on samples taken before commencement of apheresis. Individuals found to have lipemic plasma are deferred until next donation. We investigated the link between positive lipemic deferral record and low platelet CD36 expression status. RESULTS: We found four donors (0·6%) with type I CD36 deficiency (both platelets and monocytes CD36(null) ) and six (1·0%) with type II CD36 deficiency (PLT: CD36(null) , monocyte: CD36(low) ). Six CD36 genetic variants were identified, two of them were novel, all but one are found exclusively in CD36(null) and CD36(low) expressors. Subjects with CD36 genetic variants also displayed deficient or reduced CD36 on monocytes. Donors with null or low PLT CD36 expression were more likely to have a lipemic deferral record than control subjects with normal PLT CD36 expression (X(2) = 27·36, odds ratio = 2·6, 95% conference interval: 1·8-3·8, P < 0·0001). CONCLUSION: Through this study, we established a donor registry to supply CD36-negative platelets for patients in need.

Cardiac iron measurement and iron chelation therapy in patients with ß thalassaemia major: experience from Taiwan.

BACKGROUND: The aims of our study were to evaluate (i) the relationship between cardiac T2* values and cardiac complications in Asian ß-thalassaemia major (TM) patients, and (ii) the association between cardiac T2* values and other parameters currently used to predict cardiac complications as a result of transfusion iron overload. METHODS: We examined the myocardial iron loads of 88 TM patients from Taiwan with cardiac T2* magnetic resonance imaging (MRI) and assessed the correlation between cardiac T2* values and serum ferritin levels, liver iron concentration and left ventricular ejection fraction (LVEF). We also determined the predictive value of these measurements for the development of arrhythmia. RESULTS AND CONCLUSION: In our group of Taiwanese patients, the relative risk for arrhythmia was 10·36 when cardiac T2* values were less than 10 ms (compared with ≥10 ms) and 1·98 when serum ferritin levels increased >2500 ng mL(-1) (compared with ≤2500 ng mL(-1) ). Serum ferritin levels correlated with cardiac T2* values in patients with abnormal myocardial iron loads (T2* < 20 ms, r = -0·48, P = 0·004, n = 34), but LVEF (measured by echocardiography) gave no indication of excess myocardial iron deposition (r = -0·07, P = 0·52) or of the risk of developing arrhythmia.

Effect of long-term caloric restriction on DNA methylation measures of biological aging in healthy adults from the CALERIE trial.

The geroscience hypothesis proposes that therapy to slow or reverse molecular changes that occur with aging can delay or prevent multiple chronic diseases and extend healthy lifespan1-3. Caloric restriction (CR), defined as lessening caloric intake without depriving essential nutrients4, results in changes in molecular processes that have been associated with aging, including DNA methylation (DNAm)5-7, and is established to increase healthy lifespan in multiple species8,9. Here we report the results of a post hoc analysis of the influence of CR on DNAm measures of aging in blood samples from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, a randomized controlled trial in which n = 220 adults without obesity were randomized to 25% CR or ad libitum control diet for 2 yr (ref. 10). We found that CALERIE intervention slowed the pace of aging, as measured by the DunedinPACE DNAm algorithm, but did not lead to significant changes in biological age estimates measured by various DNAm clocks including PhenoAge and GrimAge. Treatment effect sizes were small. Nevertheless, modest slowing of the pace of aging can have profound effects on population health11-13. The finding that CR modified DunedinPACE in a randomized controlled trial supports the geroscience hypothesis, building on evidence from small and uncontrolled studies14-16 and contrasting with reports that biological aging may not be modifiable17. Ultimately, a conclusive test of the geroscience hypothesis will require trials with long-term follow-up to establish effects of intervention on primary healthy-aging endpoints, including incidence of chronic disease and mortality18-20.

Iron overload is associated with low anti-müllerian hormone in women with transfusion-dependent ß-thalassaemia.

OBJECTIVE: To investigate anti-müllerian hormone (AMH) as a best test of ovarian reserve in women with transfusion-dependent ß-thalassaemia, and the relationship between AMH and iron overload. DESIGN AND SETTING: A case-control study in a tertiary medical centre. POPULATION: Twenty-nine women with transfusion-dependent ß-thalassaemia and 29 healthy controls of a similar age were recruited. METHODS: Blood sampling, questionnaires and medical record reviews were used. MAIN OUTCOME MEASURES: The history of iron overload-related morbidities, haematological phenotypes, serum levels of AMH and ferritin, and hormonal profiles were analysed. RESULTS: The serum levels of AMH, luteinising hormone, and estradiol were lower in women with transfusion-dependent ß-thalassaemia than in age-matched normal controls. In women with transfusion-dependent ß-thalassaemia, the serum AMH level was significantly inversely related to the ferritin level, but not related to the presence of hypogonadotrophic hypogonadism, diabetes and haematological phenotypes. The serum ferritin level was positively associated with advanced age and the presence of hypogonadotrophic hypogonadism in the study participants. However, the inverse relationship between AMH and ferritin still exists after further adjustment for advanced age in women with transfusion-dependent ß-thalassaemia. CONCLUSIONS: The present study indicates that the serum AMH levels in women with transfusion-dependent ß-thalassaemia are lower when compared with normal healthy women of a similar age, and are significantly negatively correlated with their serum ferritin levels. This implies that ovarian function might be impaired by the chronic iron overload status in women with transfusion-dependent ß-thalassaemia.

Long-term outcome of allogeneic human leukocyte antigen-matched sibling-donor peripheral blood stem cell transplantation in leukaemia patients.

Between 1993 and July 2008, a total of 354 leukaemic patients received either allogeneic bone marrow transplantation (BMT) [n = 180] or peripheral blood stem cell transplantation (PBSCT) [n = 174] from human leukocyte antigen-matched sibling donors. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A and methotrexate. When comparing with BMT group, patients in the PBSCT group received much higher nucleated cells and CD34+ cells, and had much faster recovery of the neutrophil and platelet counts. The probability of developing acute GVHD was slightly higher in PBSCT patients (P = 0.02). The probability of chronic GVHD (cGVHD) in PBSCT was much higher in PBSCT (70 +/- 5.4%, extensive 48 +/- 6.5%) than in BMT (25 +/- 4.7%, extensive 10 +/- 3.4%; P < 0.001). Chronic GVHD was associated with long-term impairment of life quality and decreased quality-adjusted survival. In standard-risk leukaemia, use of PBSCT was associated with higher cGVHD, transplant-related mortality and a trend for decreased overall survival. The results suggest that allogeneic PBSCT is associated with high incidence of cGVHD in Chinese patients and its long-term risk and benefit remains to be defined in early stage of leukaemia.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Improved treatment results for childhood acute myeloid leukemia in Taiwan.

To improve treatment results for children with de novo acute myeloid leukemia (AML), we introduced a novel protocol, Taiwan Pediatric Oncology Group-AML-97A, for AML other than acute promyelocytic leukemia (APL), for which modified conventional protocols were used. From January 1, 1997, to December 31, 2002, 141 children younger than 17 years old with de novo AML were enrolled. In total, 117 patients with non-APL AML were treated with induction therapy of idarubicin and cytarabine (Ara-C), postremission therapy with high-dose Ara-C - containing regimens for four monthly courses, and moderate-dose therapy with idarubicin and Ara-C for four monthly courses. The first 19 patients with APL were treated with all-trans retinoic acid, idarubicin and Ara-C, with the remaining five patients receiving all-trans retinoic acid and idarubicin, followed by maintenance therapy for 2 years. Stem cell transplantation was performed in 29 patients in first remission with a similar outcome as chemotherapy alone. The remission rate in the AML-97A study was 90%, the 5-year survival 51 +/- 5.3% (s.e.) and the 5-year event-free survival 50 +/- 4.8%; for APL, these were 100%, 86 +/- 7.0, and 75 +/- 9.8%. For the whole group, the 5-year survival was 57 +/- 4.7% and the 5-year event-free survival 54 +/- 4.4%. The AML-97A regimen was well tolerated.

Accelerated DNA methylation age in adolescent girls: associations with elevated diurnal cortisol and reduced hippocampal volume.

Numerous studies have linked exposure to stress to adverse health outcomes through the effects of cortisol, a product of the stress response system, on cellular aging processes. Accelerated DNA methylation age is a promising epigenetic marker associated with stress and disease risk that may constitute a link from stress response to changes in neural structures. Specifically, elevated glucocorticoid signaling likely contributes to accelerating DNA methylation age, which may signify a maladaptive stress-related cascade that leads to hippocampal atrophy. We examined the relations among diurnal cortisol levels, DNA methylation age and hippocampal volume in a longitudinal study of 46 adolescent girls. We computed area under the curve from two daily cortisol collection periods, and calculated DNA methylation age using previously established methods based on a set of CpG sites associated with chronological age. We computed a residual score by partialling out chronological age; higher discrepancies reflect relatively accelerated DNA methylation age. We assessed hippocampal volume via T1-weighted images and automated volumetric segmentation. We found that greater diurnal cortisol production was associated with accelerated DNA methylation age, which in turn was associated with reduced left hippocampal volume. Finally, accelerated DNA methylation age significantly mediated the association between diurnal cortisol and left hippocampal volume. Thus, accelerated DNA methylation age may be an epigenetic marker linking hypothalamic-pituitary-adrenal axis dysregulation with neural structure. If these findings are replicated, the current study provides a method for advancing our understanding of mechanisms by which glucocorticoid signaling is associated with cellular aging and brain development.

Financial burden of national health insurance for treating patients with transfusion-dependent thalassemia in Taiwan.

The thalassemias are a heterogeneous group of inherited hypochromic anemias of varying severity. The mainstay of supportive treatment is regular blood transfusion accompanied by iron-chelating therapy. Hematopoietic stem cell transplantation (HSCT) provides an alternative option when curative therapy is considered. More than 400 patients in Taiwan have beta-thalassemia major or other transfusion-dependent thalassemias, and their treatment costs account for a considerable percentage of the National Health Insurance expenditure. In this report, we estimated the treatment costs of conventional therapy (regular blood transfusion accompanied by iron-chelating agents) and HSCT. The undiscounted medical cost of 20 years of follow-up (20 years from diagnosis) and the undiscounted total lifetime cost were NT$ 4 739 888 (NT$ means New Taiwan Dollars)/US$ 149 288 and NT$ 11 529 990/US$ 363 149, respectively, for patients undergoing conventional therapy, and NT$ 2 639 982/US$ 83 149 and NT$ 3 511 172/US$ 110 588, respectively, for those undergoing successful HSCT. Comparisons of treatment costs and other parameters between these two modalities can add to the information base on which policy is made by health authorities or clinicians.

Diagnostic and prognostic value of [(18)F]fluorodeoxyglucose positron emission tomography for recurrent head and neck squamous cell carcinoma.

PURPOSE: Patients with recurrent head and neck squamous cell carcinoma (HNSCC) present a diagnostic and therapeutic challenge. We evaluated the diagnostic accuracy and prognostic value of [(18)F]fluorodeoxyglucose positron emission tomography (PET) in this patient population. PATIENTS AND METHODS: We performed a retrospective review of 143 patients with previously treated HNSCC who underwent 181 PET scans at our institution from May 1996 through April 2001 to detect recurrent disease. Disease recurrence within 6 months was used as the gold standard for assessing true disease status at PET. RESULTS: With equivocal sites considered positive, the sensitivity and specificity of PET for detecting recurrence overall were 96% and 72%, respectively. PET was highly sensitive and specific at regional and distant sites. At local sites, sensitivity was high, but specificity was lower because of false-positive findings. One fifth of all false-positive PET scans occurred at sites of known inflammation or infection. The area under the curve for a receiver operator characteristic curve on the basis of standardized uptake value (SUV) was 0.882 +/- 0.025. PET interpretation, SUV, and physical examination were independent predictors of relapse-free and overall survival in a time-dependent, multivariate proportional hazards model. An increase in SUV by one unit increased the relative risk (RR) of relapse by 11% and the RR of death by 14%. A positive PET interpretation increased the RR of relapse by four-fold and the RR of death by seven-fold. CONCLUSION: PET was a highly sensitive method of detecting recurrent HNSCC and provided important prognostic information for relapse-free and overall survival.

Characterization of acute myeloid leukemia (AML) coexpressing lymphoid markers: different biologic features between T-cell antigen positive and B-cell antigen positive AML.

The clinical and biologic characteristics of acute myeloid leukemia (AML) with coexpression of lymphoid-associated antigens (Lym+ AML) were studied from 39 cases who represented 24% of 161 newly diagnosed de novo AML. Twenty-seven cases (16.8%) were positive for the expression of T-cell markers (T+ AML) and 12 (7.5%) for B-cell markers (B+ AML). Chromosomal abnormalities t(9;22)(q34;q11) and t/del(11)(q23), which were considered to be associated with acute leukemia coexpressing markers of more than one cell lineage, were detected in five and in four patients, respectively. There was no prognostic significance of B-cell or T-cell antigen expression in AML. Of 12 T+ AML cases in which cells were available for gene analysis, all showed germline configuration of immunoglobulin heavy chain and T-cell receptor beta chain genes, while seven of nine B+ AML showed rearrangements of either or both of the genes. Double labeling of the cells with myeloperoxidase and lymphoid markers demonstrated that individual blasts in all the five T+ AML tested were simultaneously expressing myeloperoxidase activity and CD7; however, most blasts in the three B+ AML studied expressed either myeloperoxidase activity or CD10, but not both. In eight of the nine T+ AML tested, the T-cell antigen-positive leukemic blasts were significantly decreased to less than 10%, after in vitro culture with the differentiation-inducing agent phorbol ester. B-cell markers remained positive (> or = 20%) on the cells in the two B+ AML who had the same study. These findings suggested that T+ AML and B+ AML might have different biologic features. Further studies on more patients are needed to clarify this point.

Characterization of Philadelphia-chromosome-positive acute leukemia by clinical, immunocytochemical, and gene analysis.

Philadelphia chromosome (Ph') was detected at presentation in 10 out of 110 patients with acute lymphoblastic leukemia (ALL) and five of 168 patients with acute myelogenous leukemia (AML). Two other ALL patients who had studies at relapse were also included in the analyses. One of the 12 Ph'-positive (Ph+) ALL patients had simultaneous expression of myeloid-associated antigen on the leukemic blasts, while all the five AML patients coexpressed markers of lymphoid cells. Double labeling of the cells with myeloperoxidase and CD10 on three Ph+ AML cases showed that most leukemic blasts expressed either myeloperoxidase activity or CD10 but not both. Cross-lineage gene rearrangements of T-cell receptor (TCR) beta-chain gene were detected in three of the eight Ph+ ALL patients tested. All the four Ph+ AML cases studied showed immunoglobulin heavy chain gene rearrangements, and three of them also had simultaneous rearrangements of TCR beta-chain gene. The results revealed that Ph+ acute leukemia in this study belonged either to ALL or mixed lineage leukemia, and none was pure AML. This finding is contrary to that of acute blast crisis of chronic myelogenous leukemia in which the majority of patients had myeloid transformation. Rearrangements of bcr were detected in four of the 10 Ph+ ALL and three of the four Ph+ AML patients tested. No significant difference was noted in the clinical or hematologic manifestations among Ph+ leukemia with or without bcr rearrangements.

Clinical and biological implications of partial tandem duplication of the MLL gene in acute myeloid leukemia without chromosomal abnormalities at 11q23.

The clinical and biological features of acute myeloid leukemia (AML) with 11q23/MLL translocations are well known, but the characteristics of AML with partial tandem duplication of the MLL gene have not been explored comprehensively. In this study, MLL duplication was analyzed, in 81 AML patients without chromosomal abnormalities at 11q23, using Southern blotting, genomic DNA polymerase chain reaction (PCR), reverse-transcription PCR and complementary DNA sequencing. Nine patients showed partial tandem duplication of the MLL gene, including eight (12%) of the 68 with normal karyotype. Seven patients showed fusion of exon 6/exon 2 (e6/e2), one, combination of differentially spliced transcripts e7/e2 and e6/e2, and the remaining one, combination of e8/e2 and e7/e2. Among the patients with normal karyotype, children aged 1 to 15 showed a trend to higher frequency of MLL duplication than other patients (2/5 or 40% vs 6/62 or 10%, P = 0.102). The patients with tandem duplication of the MLL gene had a significantly higher incidence of CD11b expression on leukemic cells than did those without in the subgroup of patients with normal karyotype (75% vs 28%, P = 0.017). There were no significant differences in the expression of lymphoid antigens or other myeloid antigens between the two groups of patients. In adults, the patients with MLL duplication had a shorter median survival time than those without (4.5 months vs 12 months, P = 0.036). In conclusion, partial tandem duplication of the MLL gene is associated with increased expression of CD11b on leukemic blasts and implicates poor prognosis in adult AML patients. The higher frequency of MLL duplication in children older than 1 year, than in other age groups, needs to be confirmed by further studies.

Characterization of acute myeloid leukemia with MLL rearrangements--no increase in the incidence of coexpression of lymphoid-associated antigens on leukemic blasts.

MLL gene rearrangements are associated with coexpression of myeloid- and lymphoid-associated antigens on leukemic blasts and a dismal outcome in acute lymphoblastic leukemia (ALL). Whether the same conditions can apply to acute myeloid leukemia (AML) is not quite clear. Rearrangements of the MLL gene were analyzed on 113 patients with newly diagnosed de novo AML in a single institution. Sixteen (14%) of them showed rearranged bands by Southern blot analysis, including three (50%) of six infants, three (14%) of 21 children between 1 and 15 years and 10 (12%) of 86 adults. MLL rearrangements were not only detected in M5 (four of 12 patients, 33%) and M4 (six of 31, 19%) subtypes but also in other non-M4-M5 AML (six of 70, 9%), including M1, M2 and M7, but not M3 subtype. Seven patients had chromosomal abnormalities involving 11q23, but nine did not. The latter comprised three (6%) of 48 patients with normal karyotype, one with t(8;21), none with t(15;17), inv(16) or t(9;22), and four (15%) of 27 with cytogenetic aberrations other than those specific structural abnormalities. In contrast to ALL, AML patients with MLL rearrangements did not tend to coexpress lymphoid- and myeloid-associated antigens simultaneously on leukemic blasts and have similar outcome as those without the gene rearrangements.

Minimally differentiated acute myeloid leukemia in Taiwan: predominantly occurs in children less than 3 years and adults between 51 and 70 years.

Acute myeloid leukemia (AML) with minimal differentiation was usually referred to as acute undifferentiated leukemia in the past. With the help of immunophenotyping, this subtype of leukemia was shown to express myeloid antigens on the blasts and was designated AML-M0 by FAB Cooperative Study Group in 1991. Among the 423 consecutive newly diagnosed de novo AML at our institution, 12 (2.8%) were of M0 subtype. The proportion of M0 in AML was higher in children than in adults (8.2% vs 1.7%). Four other M0 patients referred from outside hospitals for immunophenotyping were also included in this study. There were two peaks in age distribution of these 16 patients: less than 3 years and between 51 and 70 years, respectively. Organomegaly was more common in patients with AML-M0 than in those with other subtypes (56.3% vs 29.2%, P = 0.025). The former patients had higher incidences of CD7 and CD34 expression on the leukemic cells than the latter ones (50% vs 16.9%, P = 0.003 and 69.2% vs 37.9%, P = 0.019, respectively). The patients with AML-M0 showed more frequent clonal chromosomal abnormalities in the leukemic cells than other AML patients (83.3% vs 53.9%, P = 0.039); the same is also true for complex cytogenetic aberrations (50% vs 11. 4%, P = 0.004). Adults with AML-M0 showed a lower complete remission (CR) rate and significantly poorer survival than those with non M0-AML. However there was no significant difference in outcome between the two groups of pediatric patients. In conclusion, AML-M0 is a unique subtype of leukemia that has distinct age distribution and shows different clinical and biological characteristics from other AML. Adult patients have poor prognosis. Whether pediatric patients had better outcome than adults needs to be clarified in further studies.

Abnormal glucose tolerance in transfusion-dependent beta-thalassemic patients.

OBJECTIVE: To study the prevalence of and risk factors for abnormal glucose tolerance in transfusion-dependent beta-thalassemic patients. RESEARCH DESIGN AND METHODS: A total of 89 transfusion-dependent beta-thalassemic patients were interviewed. Diabetes was previously diagnosed in 14 of them. In the remaining 75 patients, 68 participated in an oral glucose tolerance test. Potential risk factors were identified using the independent t test, chi2 test, and Fisher's exact test. Logistic regression analysis was used to select the independent risk factors that best predicted abnormal glucose tolerance A two-tailed P value of <0.05 was considered to be statistically significant. RESULTS: The prevalence of impaired glucose tolerance was 8.5% (7 of 82) and that of diabetes was 19.5% (16 of 82). Presentation with diabetic ketoacidosis was 31.1% (5 of 16). The risk factors for abnormal glucose tolerance found in transfusion-dependent beta-thalassemic patients were serum ferritin concentration and hepatitis C infection. CONCLUSIONS: The interaction of iron overload and hepatitis C infection worsened the prognosis of thalassemic patients. Aggressive iron-chelation therapy as well as prevention and treatment of hepatitis C infection should be mandatory in managing glucose homeostasis in transfusion-dependent beta-thalassemic patients in Taiwan.

Stage-specific expression of a Plasmodium falciparum protein related to the eukaryotic mitogen-activated protein kinases.

We have identified a putative protein kinase gene from both Plasmodium falciparum cDNA and genomic DNA libraries. The nucleotide sequence contains an open-reading frame of 2646 bp, which codes for a predicted protein of 882 amino acid residues. Comparison of the predicted amino acid sequence with those in GenBank suggests that this gene codes for a protein similar to the mitogen-activated protein (MAP) kinase of other organisms. This MAP kinase-related protein, named PfMRP, contains the TDY dual phosphorylation site upstream of the highly conserved VATRWYRAPE sequence in subdomain VIII. PfMRP contains an unusually large and highly charged domain within its carboxyl-terminal segment, which includes two repetitive sequences of either a tetrapeptide or octapeptide motif. PfMRP gene is located on chromosome 14. Northern blot analysis of total RNA reveals the presence of a single mRNA transcript approximately 4.2 kb in length, which is predominantly expressed in gametocytes and gametes/zygotes.

Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum.

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression and developmental events. The acetylation state of histones is controlled by a dynamic equilibrium between activities of histone acetylase and deacetylase enzymes. Histone deacetylase (HDAC) was recently suggested to be the target of a fungus-derived antiprotozoal agent exhibiting structural similarity to known HDAC inhibitors. We have initiated a study of HDAC of human malaria parasite, Plasmodium falciparum, to evaluate its potential as the target for novel antimalarials and its role in parasite development. We have isolated HDAC1 gene from the P. falciparum genomic and cDNA libraries. The nucleotide sequence contains no intervening sequence and its open reading frame (ORF) codes for a protein of 449 amino acid residues. We have named the protein, PfHDAC1, as the sequence shows significant homology to yeast, human and other eukaryotic HDACs. Northern blot analysis of the total RNA from different asexual and sexual stages of the parasite reveals the presence of single mRNA transcript, which is predominantly expressed in mature asexual blood stages and in gametocytes. Antiserum raised against a carboxyl terminal peptide immunoprecipitated an in vitro translated P. falciparum HDAC gene product and recognized an approximately 50 kDa protein in the Triton X-100 insoluble fraction of parasites. Immunoelectron microscopy analysis showed majority of the protein localized in the nucleus of P. falciparum. To our knowledge, this is the first HDAC gene isolated from the malaria parasite.

Expression and characterisation of a Plasmodium falciparum protein containing domains homologous to sarcalumenin and a tyrosine kinase substrate, eps15.

We have identified in Plasmodium falciparum a novel gene encoding a putative bi-functional protein, termed PfPast-1, from genomic and cDNA libraries. Analysis indicated that the sequence encodes a 62 kDa protein of 529 amino acid residues with two distinctive domains: a sarcalumenin-like domain of approximately 320 amino acids at the amino half of the molecule, which shares homology to a major sarcoplasmic reticulum lumenal protein, sarcalumenin, and an eps15 homology domain of about 90 amino acids located at the carboxyl terminus. The eps15 homology domain, first identified in a tyrosine kinase substrate, eps15, and found in increasing numbers of mammalian proteins, has recently been suggested as a protein-protein interaction domain involved in intracellular sorting. Genomic sequences encoding similar proteins containing both the sarcalumenin-like and eps15 homology domains have been identified in humans and Drosophila. RNA blot analysis revealed the presence of a single messenger RNA transcript approximately 3.7 kb in size, which is expressed in all the developmental stages examined with the highest level in extracellular gametes followed by erythrocytic asexual stages, and the lowest in the gametocytes. In the attempt to define its biological function, we have expressed a full-length recombinant PfPast-1 protein in Escherichia coli. Specific immune serum directed against the recombinant protein recognised a approximately 55 kDa protein in the parasite lysate. Further characterisation of PfPast-1 may help in elucidation of its functions in P. falciparum.