Maternal dietary omega-3 fatty acid intake increases resolvin and protectin levels in the rat placenta

Placental inflammation is associated with several pregnancy disorders. Inflammation is limited by anti-inflammatory and proresolving mechanisms, the latter partly mediated by resolvins and protectins derived from omega-3 polyunsaturated fatty acids (n-3PUFA). We examined effects of dietary n-3PUFAs on levels of resolvins, protectins, and lipoxygenase (ALOX) enzymes in the rat placenta. Rats consumed standard (Std) or high n-3PUFA (Hn3) diets from day 1 of pregnancy; tissues were collected on day 17 or 22 (term = day 23). Maternal Hn3 diet increased resolvin and protectin precursors, 18R/S-HEPE (P < 0.001), and 17R/S-HDHA (P < 0.01) at both days. Resolvins (17R-RvD1 and RvD1) increased at day 22 (P < 0.001) after Hn3 consumption, coincident with higher Alox15b and Alox5 mRNA expression, while RvD2 increased at both days (P < 0.05). Protectins, PD1, and 10S,17S-DiHDHA increased over late gestation (P < 0.001), coincident with higher Alox15 mRNA expression (P < 0.001) and further increased with Hn3 diet (P < 0.05). Maternal systemic and placental proinflammatory mediators were not suppressed by Hn3 diet; systemic IL1β, placental Il1β, and Il6 mRNA expression increased marginally with Hn3 at day 22 (P < 0.001), while Ptgs1 (Cox1) expression increased both days (P < 0.05). Our data indicate that maternal n-3PUFA supplementation enhances expression of enzymes in the n-3PUFA metabolic pathway and increases placental levels of resolvins and protectins.     

Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells

In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4⁺CD25^hiCD127⁻ Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4⁺CD25⁻ counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4⁺CD25⁻ T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.     

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.     

Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling

Cellular and Molecular Neurobiology - Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type...     

Little Evidence to Support the Risk–Disturbance Hypothesis as an Explanation for Responses to Anthropogenic Noise by Pygmy Marmosets (Cebuella niveiventris) at a Tourism site in the Peruvian Amazon

The risk–disturbance hypothesis states that animals react to human stressors in the same way as they do to natural predators. Given increasing human–wildlife contact, understanding whether animals perceive anthropogenic sounds as a threat is important for assessing the long-term sustainability of wildlife tourism and proposing appropriate mitigation strategies. A study of pygmy marmoset (Cebuella niveiventris) responses to human speech found marmosets fled, decreased feeding and resting, and increased alert behaviors in response to human speech. Following this study, we investigated pygmy marmoset reactions to playbacks of different acoustic stimuli: controls (no playback, white noise and cicadas), anthropogenic noise (human speech and motorboats), and avian predators. For each playback condition, we recorded the behavior of a marmoset and looked at how the behaviors changed during and after the playback relative to behaviors before. We repeated this on ten different marmoset groups, playing each condition once to each group. The results did not replicate a previous study on the same species, at the same site, demonstrating the importance of replication in primate research, particularly when results are used to inform conservation policy. The results showed increased scanning during playbacks of the cicadas and predators compared with before the playback, and an increase in resting after playbacks of avian predators, but no evidence of behavior change in response to playbacks of human speech. There was no effect of ambient sound levels or distance between the playback source and focal animals on their behavior for all playback conditions. Although we find that noise can change the behavior of pygmy marmosets, we did not find evidence to support the risk–disturbance hypothesis.

     

Analysis of a cDNA-derived sequence of a novel mannose-binding lectin, codakine, from the tropical clam Codakia orbicularis

This work relates to the characterisation of the predominant gill protein of the white clam, Codakia orbicularis (Linné, 1758), which harbours endosymbiotic sulphur-oxidising chemoautotrophic bacteria. Total RNA was extracted from the clam to perform 3'rapid amplification of cDNA ends (3'RACE) using degenerate oligonucleotides prepared from a partial sequence of a predominant protein of about 14kDa, termed codakine. The partial peptide sequence was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Edman degradation. Clones isolated from the cDNA library and containing the gene of interest were used in polymerase chain reaction (PCR). The PCR and RACE-PCR products were sequenced to determine the entire coding DNA sequence of codakine. BLAST analysis revealed about 23% to 29% sequence identity between codakine and various animal C-type lectins that are often involved in symbiosis and immune defences. Codakine also contains the motifs and domains of Ca(2+)-dependent C-type lectins, and in particular the tripeptide EPN motif frequently found in mannose-binding lectins (MBLs). Analysis of the protein by affinity chromatography on a mannose-agarose column is consistent with the findings that codakine is a dimeric Ca(2+)-dependent MBL of about 29kDa. Based on the present results, it is hypothesised that this novel C. orbicularis gill protein is involved in the recognition of symbiotic and pathogenic bacteria.     

Assessing the role of the active-site cysteine ligand in the superoxide reductase from Desulfoarculus baarsii

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of O(2)(.) to H(2)O(2), providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe(2+) center in a [His(4) Cys(1)] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu(114), is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu(114) carboxylic lateral chain affects the strength of the S-Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S-Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H(2)O(2) by a specific strengthening of the Fe-O bond. A fine tuning of the strength of the S-Fe bond by the presence of Glu(114) appears to be an essential factor for both the strength of the Fe-O bond and the pK(a) value of the Fe(3+)-peroxo intermediate species to form the reaction product H(2)O(2).     

Kinetic and mutational analyses of the major cytosolic exopolyphosphatase from Saccharomyces cerevisiae

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.     

The role of p63 in germ cell apoptosis in the developing testis

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.