Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism

Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction.

INTRODUCTION: Transport of glucose from maternal blood across the placental trophoblastic tissue barrier is critical to sustain fetal growth. The mechanism by which GLUTs are regulated in trophoblasts in response to ischemic hypoxia encountered with intrauterine growth restriction (IUGR) has not been suitably investigated. OBJECTIVE: To investigate placental expression of GLUT1, GLUT3 and GLUT4 and possible mechanisms of GLUT regulation in idiopathic IUGR. METHODS: We analyzed clinical, biochemical and histological data from placentas collected from women affected by idiopathic full-term IUGR (n = 10) and gestational age-matched healthy controls (n = 10). RESULTS: We found increased GLUT3 protein expression in the trophoblast (cytotrophoblast greater than syncytiotrophoblast) on the maternal aspect of the placenta in IUGR compared to normal placenta, but no differences in GLUT1 or GLUT4 were found. No differential methylation of the GLUT3 promoter between normal and IUGR placentas was observed. Increased GLUT3 expression was associated with an increased nuclear concentration of HIF-1α, suggesting hypoxia may play a role in the up-regulation of GLUT3. DISCUSSION: Further studies are needed to elucidate whether increased GLUT3 expression in IUGR is a marker for defective villous maturation or an adaptive response of the trophoblast in response to chronic hypoxia. CONCLUSIONS: Patients with IUGR have increased trophoblast expression of GLUT3, as found under the low-oxygen conditions of the first trimester.

Feasibility of administering zoledronic acid in palliative patients being cared for in the community: results of a pilot study.

Tumour-induced hypercalcemia (TIH) and pain from bone metastases are common complications of advanced malignancy and have a significant negative impact on quality of life. Many cancer patients in the advanced stages of their palliative illness prefer to avoid hospitalization and to receive their care in the community setting. This small open-label prospective pilot study explored the feasibility of administering zoledronic acid intravenously in the community setting (home and residential hospices). It enrolled a convenience sample of 12 patients with advanced cancer and TIH (n = 7), malignant bone pain (n = 3), or TIH and malignant bone pain (n = 2). The mean duration of infusion was 15 minutes (range: 14-30 minutes). The total nursing time required was 95 minutes, and the mean total cost, including nursing time, travel time, and drug costs was $708.97 per infusion. This cost was compared with costs for clodronate and pamidronate ($402.52 and $406.12 respectively). Calcium fell from a mean of 2.97 mmol/L on day 0 to 2.63 mmol/L on day 4 and to 2.54 mmol/L on day 10. Delirium resolved in 2 of 5 patients with TIH-associated delirium. Intravenous zoledronic acid administered in the community to palliative patients at the end of life is feasible and safe, and the short duration of infusion offers advantages to patients and nursing resources alike. The higher cost of zoledronic acid per infusion may be offset by the advantage of its short infusion time.

The effect of strength training combined with bisphosphonate (etidronate) therapy on bone mineral, lean tissue, and fat mass in postmenopausal women.

The combined and separate effects of exercise training and bisphosphonate (etidronate) therapy on bone mineral in postmenopausal women were compared. Forty-eight postmenopausal women were randomly assigned (double blind) to groups that took intermittent cyclical etidronate; performed strength training (3 d/week) and received matched placebo; combined strength training with etidronate; or took placebo and served as nonexercising controls. Bone mineral, lean tissue, and fat mass were assessed by dual-energy X-ray absorptiometry before and after 12 months of intervention. After removal of outlier results, changes in bone mineral density (BMD) of the lumbar spine and bone mineral content (BMC) of the whole body were greater in the subjects given etidronate (+2.5 and +1.4%, respectively) compared with placebo (-0.32 and 0%, respectively) (p < 0.05), while exercise had no effect. There was no effect of etidronate or exercise on the proximal femur and there was no interaction between exercise and etidronate at any bone site. Exercise training resulted in significantly greater increases in muscular strength and lean tissue mass and greater loss of fat mass compared with controls. We conclude that etidronate significantly increases lumbar spine BMD and whole-body BMC and that strength training has no additional effect. Strength training favourably affects body composition and muscular strength, which may be important for prevention of falls.

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

Formation and in situ sizing of gamma-Fe2O3 nanoparticles in a microwave flow reactor.

Nanocrystalline gamma-Fe2O3 particles were produced in a microwave flow reactor. The reaction of iron pentacarbonyl [Fe(CO)5] with the plasma gases Ar/O2 to form nanosized particles was followed by in situ particle mass spectrometry. The particle mass spectrometer combines a nonintrusive sampling technique with a calibration-free mass determination. The influence of process parameters like microwave power, precursor concentration, and pressure on the particle size was studied. The results reveal a mean particle diameter in the range of 4-5 nm with a slight dependence on the process parameter. The geometric standard deviation of the measured size distribution was always between 1.1 and 1.2.

CIITA leucine-rich repeats control nuclear localization, in vivo recruitment to the major histocompatibility complex (MHC) class II enhanceosome, and MHC class II gene transactivation.

The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.

A monomeric GTPase-negative MxA mutant with antiviral activity.

MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.

MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures.

MxA protein is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses, including influenza viruses and bunyaviruses. Studies on MxA transgenic mice have shown that MxA is a powerful antiviral agent in vivo. It has been suggested that this cellular protein also protects humans from viral disease, but the mechanism(s) by which MxA exerts its antiviral action is still poorly understood. Using an in vitro cosedimentation assay, we now demonstrate that MxA tightly interacts with components of the ribonucleoprotein complex of Thogoto virus, an influenza-like virus transmitted by ticks. This assay demonstrates for the first time a physical interaction between MxA GTPase and a viral target structure. It is based on three elements, namely, highly active MxA GTPases as effector molecules, viral ribonucleoprotein particles as viral targets, and GTPgammaS as a stabilizing factor. Furthermore, using a simple nuclear translocation assay, we show that human MxA protein forms oligomers in vivo. This assay provides a stringent test for tight association of partner molecules in intact mammalian cells. It not only will be useful for studying physical interactions of MxA with partner molecules, but may also be applicable to other studies on protein-protein interactions in living cells.

A Canadian hospital-based HIV/hepatitis C look-back notification program.

OBJECTIVE: To describe the process used to notify pediatric patients who received transfusions of blood or blood products at our institution before donor blood was routinely screened for antibodies to HIV (1985) and hepatitis C virus (1990), and to evaluate the effectiveness of the notification program. DESIGN: Patients who had received transfusions were identified through the hospital's medical records and the records from the Transfusion Medicine Laboratory. Patients were contacted by registered mail to provide notification of transfusion. A questionnaire was included with the notification to obtain information about the patient's awareness of the transfusion and whether he or she had undergone or planned to undergo testing for HIV and hepatitis C virus. SETTING: Tertiary care university-affiliated teaching hospital in Hamilton, Ont. PATIENTS: Patients 16 years of age or younger who had received blood products between February 1978 and November 1985. Patients who had received only albumin or immune serum globulin were not included as these products were not associated with viral transmission in Canada. RESULTS: Notification letters were sent to 1546 patients. Of these letters 522 (33.8%) were returned undelivered. Of the 1024 patients contacted 493 (48.1%) responded to the questionnaire, of whom 157 (31.8%) were not aware of their transfusion. A total of 130 (26.4%) of the respondents had already undergone testing for HIV, and 342 (69.4%) indicated that they would undergo such testing as a result of the notification. In contrast, only 30 (6.3%) of 474 respondents had undergone testing for hepatitis C virus, but 425 (89.7%) indicated that they would undergo such testing. Overall, the patients' response to the notification was neutral or positive; however, a number of patients expressed dissatisfaction and anxiety. CONCLUSIONS: The high proportion of patients who were unaware that they had undergone transfusion and who decided to undergo testing for HIV and hepatitis C virus as a result of notification supports the use of notification programs such as this one.

A single STAT recruitment module in a chimeric cytokine receptor complex is sufficient for STAT activation.

We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor alpha and beta chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the alpha and the beta chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates. We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.

A structural model for fidelity in transcription.

Distances between the metal ions bound to the product terminus i site and the substrate i + 1 site of Escherichia coli RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship. The metal bound to the substrate at the i + 1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the distance to base and ribose protons changes. The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position. In this scheme a metal-triphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.

Child psychiatry education in early child development: description of a training program.

Career training in child psychiatry, though provided from a developmental perspective, often lacks opportunity for directly observing normally developing infants. This paper describes a training program in which child psychiatry fellows, in collaboration with residents in pediatrics, follow a young couple and their first child from the third trimester of pregnancy through infancy and early childhood. Case illustrations and discussion of approximately 10 years' experience demonstrate how the program can enhance understanding of normal child development, development of the nuclear family, liaison between child psychiatrist and pediatrician and early detection of psychopathology in infants.

Families in the treatment of alcoholism.

A review of the literature on family treatment of alcoholism suggests that although there is no single definition of family therapy, nor one single theory behind it, there is agreement that family therapy can be beneficial for both the alcoholic and the family.