Associative learning in wild Anastrepha obliqua females (Diptera, Tephritidae) related to a protein source

The aim of the present study was to determine whether wild adult Anastrepha obliqua (Macquart, 1835) females are able to associate a compound (quinine sulphate - QS) not related to their habitual diet with a protein-enriched food. Females were first fed on diets based on brewer yeast and sucrose containing or not QS. The groups were then allowed to choose between their original diets and a diet with or without QS, depending on the previous treatment, and between a diet based on agar and a diet containing agar and QS. When the nutritional value of the diets was adequate, the females did not show any preference for the diet with or without QS. With respect to the agar diet and the agar + QS diet, females previously fed on a nutritive diet containing QS preferred the diet containing QS, indicating an association between the compound and the nutritional value of the diet. The importance of this behavioral strategy is discussed.

Seasonal variation of the effect of high-carbohydrate and high-protein diets on the intermediate metabolism of Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) maintained in the laboratory

The goal of this study was to evaluate the effect of a high-carbohydrate diet (HC) and a high-protein diet (HP) on the metabolism of the crayfish Parastacus brasiliensis (Von Martens, 1869), collected in different seasons and maintained in the laboratory for 15 days. Crayfish were collected monthly from January 2002 to January 2004 at São Francisco de Paula, Southern Brazil, in Guarapirá stream. In the laboratory, the animals were kept submerged in aquariums under controlled conditions. They were fed ad libitum, for 15 days with either a HC or HP diet. At the end of this period, haemolymph samples were collected, as were hepatopancreas, gills, and abdominal muscle that were removed for determination of glycogen, free glucose, lipids, and triglycerides. The haemolymph samples were used for determination of glucose, proteins, lipids, and triglycerides. Statistical analysis (ANOVA) revealed significant seasonal differences in biochemical composition in crayfish maintained on HC or HP diets. Independent of the diets offered to the animals and the controlled conditions for 15 days, the indications of seasonality were unchanged. The observed changes seemed to be related to the reproductive period. Moreover, the HC diet increased all energy reserves in adult parastacids, which may aid in reproduction.

Inhibitory effect of cyclophosphamide on the biosynthesis of a perchloro-soluble protein fraction of ehrlich ascites carcinoma cells

The perchloro-soluble mucroptotein fraction was determined in the cells of Ehrlich ascites carcinoma on the 10th and 12th days post-inoculation of the tumor. After 3 days of a single subcutaneous dose of cyclophosphamide (200 mg/kg) the mucoprotein levels were found considerable lower. This difference was highly significant statistically.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes.

The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L.

MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Fecal calprotectin more accurately reflects endoscopic activity of ulcerative colitis than the Lichtiger Index, C-reactive protein, platelets, hemoglobin, and blood leukocytes.

BACKGROUND: The correlation between noninvasive markers with endoscopic activity according to the modified Baron Index in patients with ulcerative colitis (UC) is unknown. We aimed to evaluate the correlation between endoscopic activity and fecal calprotectin (FC), C-reactive protein (CRP), hemoglobin, platelets, blood leukocytes, and the Lichtiger Index (clinical score). METHODS: UC patients undergoing complete colonoscopy were prospectively enrolled and scored clinically and endoscopically. Samples from feces and blood were analyzed in UC patients and controls. RESULTS: We enrolled 228 UC patients and 52 healthy controls. Endoscopic disease activity correlated best with FC (Spearman's rank correlation coefficient r = 0.821), followed by the Lichtiger Index (r = 0.682), CRP (r = 0.556), platelets (r = 0.488), blood leukocytes (r = 0.401), and hemoglobin (r = -0.388). FC was the only marker that could discriminate between different grades of endoscopic activity (grade 0, 16 [10-30] μg/g; grade 1, 35 [25-48] μg/g; grade 2, 102 [44-159] μg/g; grade 3, 235 [176-319] μg/g; grade 4, 611 [406-868] μg/g; P < 0.001 for discriminating the different grades). FC with a cutoff of 57 μg/g had a sensitivity of 91% and a specificity of 90% to detect endoscopically active disease (modified Baron Index ≥ 2). CONCLUSIONS: FC correlated better with endoscopic disease activity than clinical activity, CRP, platelets, hemoglobin, and blood leukocytes. The strong correlation with endoscopic disease activity suggests that FC represents a useful biomarker for noninvasive monitoring of disease activity in UC patients.

Theoretical study on receptor-G protein recognition: new insights into the mechanisms of the alpha1b-adrenergic recptor activation

This work compares the structural/dynamics features of the wild-type alb-adrenergic receptor (AR) with those of the D142A active mutant and the agonist-bound state. The two active receptor forms were compared in their isolated states as well as in their ability to form homodimers and to recognize the G alpha q beta 1 gamma 2 heterotrimer. The analysis of the isolated structures revealed that, although the mutation- and agonist-induced active states of the alpha 1b-AR are different, they, however, share several structural peculiarities including (a) the release of some constraining interactions found in the wild-type receptor and (b) the opening of a cytosolic crevice formed by the second and third intracellular loops and the cytosolic extensions of helices 5 and 6. Accordingly, also their tendency to form homodimers shows commonalties and differences. In fact, in both the active receptor forms, helix 6 plays a crucial role in mediating homodimerization. However, the homodimeric models result from different interhelical assemblies. On the same line of evidence, in both of the active receptor forms, the cytosolic opened crevice recognizes similar domains on the G protein. However, the docking solutions are differently populated and the receptor-G protein preorientation models suggest that the final complexes should be characterized by different interaction patterns.

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax

The cellular immune response to the circumsporozoite (CS) protein of plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequenceof the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen specific in vitro proliferative responseto the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative reponse when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, withinthe major surface antigen of P. vivax sporozoites, of epitopes capble to induce proliferation of human PBMC.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.