The-202 A Allele of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Promoter Polymorphism Is Associated with Higher IGFBP-3 Serum Levels and Better Growth Response to Growth Hormone Treatment in Patients with Severe Growth Hormone Deficiency

Context: Genetic factors that influence the response to recombinant human GH (rhGH) therapy remain mostly unknown. To date, only the GH receptor gene has been investigated. Objective: The aim of the study was to assess the influence of a polymorphism in the IGF-binding protein-3 (IGFBP-3) promoter region (-202 A/C) on circulating IGFBP-3 levels and growth response to rhGH therapy in children with GH deficiency (GHD). Design and Patients: -202 A/C IGFBP3 genotyping (rs2854744) was correlated with data of 71 children with severe GHD who remained prepubertal during the first year of rhGH treatment. Main Outcome Measures: We measured IGFBP-3 levels and first year growth velocity (GV) during rhGH treatment. Results: Clinical and laboratory data at the start of treatment were indistinguishable among patients with different -202 A/C IGFBP3 genotypes. Despite similar rhGH doses, patients homozygous for the A allele presented higher IGFBP-3 SD score levels and higher mean GV in the first year of rhGH treatment than patients with AC or CC genotypes (first year GV, AA = 13.0 +/- 2.1 cm/yr, AC = 11.4 +/- 2.5 cm/yr, and CC = 10.8 +/- 1.9 cm/yr; P = 0.016). Multiple linear regression analyses demonstrated that the influence of -202 A/C IGFBP3 genotype on IGFBP-3 levels and GV during the first year of rhGH treatment was independent of other variables. Conclusion: The -202 A allele of IGFBP3 promoter region is associated with increased IGFBP-3 levels and GV during rhGH treatment in prepubertal GHD children. (J Clin Endocrinol Metab 94: 588-595, 2009)

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

NEONATAL HANDLING AND THE MATERNAL ODOR PREFERENCE IN RAT PUPS: INVOLVEMENT OF MONOAMINES AND CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN PATHWAY IN THE OLFACTORY BULB

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Distinct subsets of hypothalamic genes are modulated by two different thermogenesis-inducing stimuli

Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-D-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role ill Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey ill the Brazilian Amazon Basin. Despite Continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the Population was reexamined 12 months later. Antibodies from 16 of 50 (360%) Subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by Multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri

In Xanthomonas axonopodis pv. citri (Xac or X citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala(38) and Ser(151), are shown to be part of the ligand-binding pocket. (c) 2007 Elsevier B.V All rights reserved.

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP(II)), we performed in vitro assays with mammalian cells expressing DBP(II) sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP(II) are short-lived and biased towards a specific allele.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Role of Rab27 in synaptic transmission at the squid giant synapse

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca2+ current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca2+-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15: 17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer ""stem"" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), Fc gamma RIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) possibly through a methylation-dependent mechanism, indicating that C/EBP alpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. (Blood. 2009; 114: 5415-5425)

Ric-8A, a G alpha protein guanine nucleotide exchange factor potentiates taste receptor signaling

Taste receptors for sweet, bitter and umami tastants are G-protein-coupled receptors (GPCRs). While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS), RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs) are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of G alpha subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with G alpha-gustducin and G alpha i2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Tick saliva inhibits the chemotactic function of MIP-1 alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5

Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3 beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1 alpha, while it did not affect RANTES, MIP-1 beta and MIP-3 beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48 h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.