Reptin and pontin antagonistically regulate heart growth in zebrafish embryos

Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway.     

Multiple mechanisms compensate to enhance tumor-protective CD8(+) T cell response in the long-term despite poor CD8(+) T cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen

We evaluated CD8(+) T cell responses against the dominant CTL epitope, OVA(257-264), expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8(+) T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8(+) T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4(+) T cells. CD8(+) T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8(+) T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

Association of Grb7 with phosphoinositides and its role in the regulation of cell migration

Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.     

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.     

A genomewide linkage scan for quantitative-trait loci for obesity phenotypes

Obesity is an increasingly serious health problem in the world. Body mass index (BMI), percentage fat mass, and body fat mass are important indices of obesity. For a sample of pedigrees that contains >10,000 relative pairs (including 1,249 sib pairs) that are useful for linkage analyses, we performed a whole-genome linkage scan, using 380 microsatellite markers to identify genomic regions that may contain quantitative-trait loci (QTLs) for obesity. Each pedigree was ascertained through a proband who has extremely low bone mass, which translates into a low BMI. A major QTL for BMI was identified on 2q14 near the marker D2S347 with a LOD score of 4.04 in two-point analysis and a maximum LOD score (MLS) of 4.44 in multipoint analysis. The genomic region near 2q14 also achieved an MLS >2.0 for percentage of fat mass and body fat mass. For the putative QTL on 2q14, as much as 28.2% of BMI variation (after adjustment for age and sex) may be attributable to this locus. In addition, several other genomic regions that may contain obesity-related QTLs are suggested. For example, 1p36 near the marker D1S468 may contain a QTL for BMI variation, with a LOD score of 2.75 in two-point analysis and an MLS of 2.09 in multipoint analysis. The genomic regions identified in this and earlier reports are compared for further exploration in extension studies that use larger samples and/or denser markers for confirmation and fine-mapping studies, to eventually identify major functional genes involved in obesity.     

Dynamic modification of cortical orientation tuning mediated by recurrent connections

Receptive field properties of visual cortical neurons depend on the spatiotemporal context within which the stimuli are presented. We have examined the temporal context dependence of cortical orientation tuning using dynamic visual stimuli with rapidly changing orientations. We found that tuning to the orientation of the test stimulus depended on a briefly presented preceding stimulus, with the preferred orientation shifting away from the preceding orientation. Analyses of the spatial-phase dependence of the shift showed that the effect cannot be explained by purely feedforward mechanisms, but can be accounted for by activity-dependent changes in the recurrent interactions between different orientation columns. Thus, short-term plasticity of the intracortical circuit can mediate dynamic modification of orientation tuning, which may be important for efficient visual coding.     

Characterization of rhodamine conjugated Agiotensin II peptide: synthesis,analysis and receptor binding and internalization

The results in this study show that the rhodamine fluorophore can be specifically conjugated to Angiotensin II at Lys3 residue (substituted for a Val) without altering the biological activity of the parent compound. The conjugated peptide was characterized using HPLC, mass spectrometry, and N-terminal sequencing. The rhodamine-Angiotensin II binds effectively to AT1 receptor and gets internalized in clathrin coated vesicles by endocytosis. These results clearly suggest the usefulness of fluorophore-conjugated peptides in studies such as, ligand-receptor binding, and ligand-receptor complex internalization, for drug delivery using cell receptors and as an alternative to peptide hormone radioimmunoassays.     

Crystallization and preliminary X-ray diffraction analysis of FKBP12 complexed with a novel neurotrophic ligand

A novel neurotrophic ligand, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester, has been complexed with FKBP12 and crystallized using the hanging-drop vapor-diffusion method. Crystals belong to P2(1) space group, with unit cell parameters a=41.2, b=29.6, c=41.5 A, beta=114.0 degrees. The crystals diffract to 1.8 A resolution limit.