Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a flipflop phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with log. CRP. concentration across a 0.5-50. nM range and associated with a limit of detection of 176. pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed. © 2012.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1-/- mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach. Copyright © 2013 by The American Association of Immunologists, Inc.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate carcinoma

Aims: To investigate the prognostic value of expression levels of the genes STEAP1 and STEAP2, and of STEAP1 protein, in prostate carcinomas (PCa). Methods and results: STEAP1 and STEAP2 transcript levels were evaluated by RT-qPCR in samples from 35 PCa, 24 adjacent non-neoplastic prostate (AdjP) tissues, five cases of benign prostatic hyperplasia (BPH), and two histologically normal prostates (N). STEAP1 expression was assessed by immunohistochemistry in samples from 198 PCa, 76 AdjP, 22 BPH, and two N. The findings were compared with clinical and pathological parameters and patient outcome. STEAP1 and STEAP2 transcript analysis showed no differences between the groups tested. Although not significant, higher STEAP1 mRNA levels were detected in tumours with high Gleason scores and in patients who presented with biochemical recurrence (BCR). STEAP1 overexpression was detected in PCa, and was significantly associated with high-grade Gleason scores, seminal vesicle invasion, BCR, and worse outcome (metastasis or PCa-specific death). STEAP1 overexpression was significantly associated with shorter BCR-free survival. Multivariate analysis revealed that STEAP1 is an independent marker for BCR. Conclusions: These findings provide evidence that STEAP1 is a biomarker of worse prognosis in PCa patients. © 2013 John Wiley & Sons Ltd.

High levels of heat shock protein 70 are associated with pro-inflammatory cytokines and may differentiate early- from late-onset preeclampsia

Preeclampsia (PE), a specific syndrome of pregnancy, can be classified into early and late onset, depending on whether clinical manifestations occur before or after 34 weeks' gestation. We determined whether plasma concentrations of Hsp60 and Hsp70 were related to circulating cytokine levels, as well as kidney and liver functions, in early- and late-onset PE. Two hundred and thirty-seven preeclamptic women (95 with early- and 142 with late-onset PE) were evaluated. Plasma levels of Hsp60, Hsp70, and their specific antibodies, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-10, IL-12, and soluble TNF-α-receptor I (sTNFRI) concentrations, were determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of Hsp70, TNF-α, IL-1β, IL-12, and sTNFRI were significantly elevated in patients with early-onset PE compared with women with late-onset PE; IL-10 levels were significantly lower in the early-onset PE group. Concentrations of urea, uric acid, proteinuria, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were also significantly higher in early-onset PE. The percentage of infants with intrauterine growth restriction was also significantly higher in women with early-onset PE. There were positive correlations between Hsp70 levels and TNF-α, TNFRI, IL-1β, IL-12, GOT, GPT, LDH, and uric acid concentrations in early-onset PE group. Thus, early-onset PE was associated with greater maternal and fetal impairment. There are differences in pathophysiology between early- and late-onset PE, highlighting by the difference in Hsp70 levels. © 2013 Elsevier B.V. All rights reserved.

High-fiber diets with reduced crude protein for commercial layers

This study aimed at evaluating diets containing different fiber sources and two crude protein levels on the performance, egg quality, and nitrogen metabolism of commercial layers. In total, 392 48-wk-old Isa Brown layers were distributed according to a completely randomized experimental design in a 3x2+1 (control) factorial arrangement, resulting in seven treatments with seven replicates of eight birds each. Treatments consisted of three fiber feedstuffs (cottonseed hulls, soybean hulls, and rice hulls) and two dietary crude protein levels (12% and 16%). Cottonseed hulls associated with the high crude protein level (16%) resulted in the worst feed conversion ratio per dozen eggs. Diets with 16% crude protein resulted in the highest feed intake, egg production, egg weight, egg mass values, and improved feed conversion ratio (kg eggs/kg feed). The dietary inclusion of soybean hulls determined low yolk pigmentation, and of rice hulls, low egg specific gravity. The 16% crude protein diet with rice hulls promoted the best feed conversion ratio. Hens fed the reference diet presented higher egg mass and better feed conversion ratio per kg eggs and per dozen eggs. Hens fed the diets with low crude protein level (12%) had reduced nitrogen excretion, but presented worse egg production.

Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hypusine Modification of the Ribosome-binding Protein eIF5A, a Target for New Anti-Inflammatory Drugs: Understanding the Action of the Inhibitor GC7 on a Murine Macrophage Cell Line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

3D osteoarthritic changes in TMJ condylar morphology correlates with specific systemic and local biomarkers of disease

Objective: To assess 3D morphological variations and local and systemic biomarker profiles in subjects with a diagnosis of temporomandibular joint osteoarthritis (TMJ OA).Design: Twenty-eight patients with long-term TMJ OA (39.9 +/- 16 years), 12 patients at initial diagnosis of OA (47.4 +/- 16.1 years), and 12 healthy controls (41.8 +/- 12.2 years) were recruited. All patients were female and had cone beam CT scans taken. TMJ arthrocentesis and venipuncture were performed on 12 OA and 12 age-matched healthy controls. Serum and synovial fluid levels of 50 biomarkers of arthritic inflammation were quantified by protein microarrays. Shape Analysis MANCOVA tested statistical correlations between biomarker levels and variations in condylar morphology.Results: Compared with healthy controls, the OA average condyle was significantly smaller in all dimensions except its anterior surface, with areas indicative of bone resorption along the articular surface, particularly in the lateral pole. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were significantly correlated with bone apposition of the condylar anterior surface. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGF beta b1, IFN gamma g, TNF alpha a, IL-1 alpha a, and IL-6 were significantly correlated with flattening of the lateral pole. Expression levels of ANG were significantly correlated with the articular morphology in healthy controls.Conclusions: Bone resorption at the articular surface, particularly at the lateral pole was statistically significant at initial diagnosis of TMJ OA. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were correlated with bone apposition. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGF beta 1, IFN gamma, TNF alpha, IL-1 alpha, and IL-6 were correlated with bone resorption. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

New ligands of the cellular retinoic acid-binding protein 2 (CRABP2) suggest a role for this protein in chromatin remodeling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of aging and maternal protein restriction on the muscle fibers morphology and neuromuscular junctions of rats after nutritional recovery

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)