Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2^LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2^LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2^LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.     

Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.     

Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.     

Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines

p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.     

Differential requirement for pulsatile LH during the follicular phase and exposure to the preovulatory LH surge for oocyte fertilization and embryo development in cattle

The requirement for pulsatile LH and the LH surge for the acquisition of oocyte fertilizing potential and embryo developmental competency was examined in Zebu heifers. Follicular growth was superstimulated using the GnRH agonist-LH protocol in which pulsatile LH and the preovulatory LH surge are blocked. In experiment 1, heifers were assigned on Day 7 of the estrous cycle to receive: group 1A (n = 5), 1.5 mg norgestomet (NOR) implant; group 1B (n = 5), GnRH agonist implant. Follicular growth was superstimulated with 2x daily injections of FSH from Day 10 (a.m.) to Day 13 (p.m.), with PGF2alpha injection on Day 12 (a.m.). Heifers were ovariectomized on Day 15 (a.m.) and oocytes were placed immediately into fertilization, without 24 h maturation. Respective cleavage and blastocyst development rates were: group 1A, 0/64 oocytes (0%) and 0/64 (0%); group 1B, 34/70 oocytes (48.6%) and 2/70 (2.9%). In experiment 2, heifers were assigned on Day 7 of the estrous cycle to receive: group 2A (n = 10), 1.5 mg NOR implant; group 2B (n = 10), GnRH agonist implant; group 2C (n = 10), GnRH agonist implant. Follicular growth was superstimulated as in experiment 1 above. Heifers in groups 2A and 2B received an injection of 25 mg LH on Day 14 (p.m.) and all heifers were ovariectomized on Day 15 (a.m.); oocytes were placed immediately into fertilization without 24 h maturation. Cleavage rates were similar for heifers in group 2A (84/175 oocytes, 48.0%), group 2B (61/112 oocytes, 54.5%) and group 2C (69/163, 42.3%). Blastocyst development rates were similar for heifers in group 2A (22/175 oocytes, 12.6%) and group 2B (25/112 oocytes, 22.3%) and lower (P < 0.05) for heifers in group 2C (9/163 oocytes, 5.5%). Oocytes obtained from heifers treated with GnRH agonist, without injection of exogenous LH, underwent cleavage indicating that neither pulsatile LH nor the preovulatory LH surge are obligatory for nuclear maturation in cattle oocytes. Exposure to a surge-like increase in plasma LH increased embryo developmental competency indicating that the preovulatory LH surge promotes cytoplasmic maturation. The findings have important implications for controlling the in vivo maturation of oocytes before in vitro procedures including nuclear transfer.