Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue

Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Noninvasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in?vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively active CXCR4 mutation in zebrafish warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.     

Viscoelastic properties of bovine orbital connective tissue and fat: constitutive models

Reported mechanical properties of orbital connective tissue and fat have been too sparse to model strain–stress relationships underlying biomechanical interactions in strabismus. We performed rheological tests to develop a multi-mode upper convected Maxwell (UCM) model of these tissues under shear loading. From 20 fresh bovine orbits, 30 samples of connective tissue were taken from rectus pulley regions and 30 samples of fatty tissues from the posterior orbit. Additional samples were defatted to determine connective tissue weight proportion, which was verified histologically. Mechanical testing in shear employed a triborheometer to perform: strain sweeps at 0.5–2.0 Hz; shear stress relaxation with 1% strain; viscometry at 0.01−0.5 s⁻¹ strain rate; and shear oscillation at 1% strain. Average connective tissue weight proportion was 98% for predominantly connective tissue and 76% for fatty tissue. Connective tissue specimens reached a long-term relaxation modulus of 668 Pa after 1,500 s, while corresponding values for fatty tissue specimens were 290 Pa and 1,100 s. Shear stress magnitude for connective tissue exceeded that of fatty tissue by five-fold. Based on these data, we developed a multi-mode UCM model with variable viscosities and time constants, and a damped hyperelastic response that accurately described measured properties of both connective and fatty tissues. Model parameters differed significantly between the two tissues. Viscoelastic properties of predominantly connective orbital tissues under shear loading differ markedly from properties of orbital fat, but both are accurately reflected using UCM models. These viscoelastic models will facilitate realistic global modeling of EOM behavior in binocular alignment and strabismus.     

Grazing impact of heterotrophic dinoflagellates and ciliates on common red-tide euglenophyte Eutreptiella gymnastica in Masan Bay, Korea

The euglenophyte Eutreptiella gymnastica is a common red tide causative species. However, there have been no studies on the grazing impact of heterotrophic protists on this species. To investigate the grazing impact of heterotrophic protists on E. gymnastica, we measured daily the abundances of E. gymnastica and co-occurring potential heterotrophic protistan grazers in Masan Bay, Korea, in August 2004 when an E. gymnastica red tide occurred. In addition, we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, Oxyrrhis marina, Pfiesteria piscicida, Polykrikos kofoidii, Protoperidinium bipes, and Stoeckeria algicida and the naked ciliates Strobilidium sp. (30–40 μm in cell length) and Strombidinopsis sp. (70–100 μm in cell length) were able to feed on E. gymnastica. We also measured their growth and ingestion rates on E. gymnastica as a function of prey concentration. Finally, we calculated the grazing coefficients by combining field data on the abundance of the heterotrophic dinoflagellate and ciliate grazers and co-occurring E. gymnastica with laboratory data on ingestion rates obtained in this study. The maximum abundance of E. gymnastica in Masan Bay in August, 2004 was 7575 cells ml⁻¹, while those of Gyrodinium spp., P. kofoidii, P. bipes, the naked ciliates (≤50 μm in cell length), and naked ciliates (>50 μm in cell length) were 50, 9, 58, 32, and 3 cells ml⁻¹, respectively. The maximum growth rate of G. dominans on E. gymnastica (1.13 d⁻¹) was higher than that of O. marina (0.81 d⁻¹) or P. bipes (0.77 d⁻¹). However, E. gymnastica did not support positive growth of P. kofoidii, Strobilidium sp., and Strombidinopsis sp. (−0.04 ∼ −2.8 d⁻¹). The maximum ingestion rates of G. dominans, P. kofoidii, P. bipes, O. marina, and Strobilidium sp. on E. gymnastica (2.1–2.7 ng C predator⁻¹ d⁻¹) were similar, but they were much lower than that of Strombidinopsis sp. (156 ng C predator⁻¹ d⁻¹). The calculated grazing coefficients for P. bipes, small heterotrophic Gyrodinium spp. (25–35 μm in cell length), naked ciliates (≤50 μm in cell length), P. kofoidii, and naked ciliates (>50 μm in cell length) on E. gymnastica were up to 0.77, 0.61, 0.22, 0.07 and 0.03 d⁻¹, respectively (i.e., up to 54%, 46%, 20%, 7%, and 3% of E. gymnastica populations were removed by the population of each of these heterotrophic protistan grazers in 1 d, respectively). The results of the present study suggest that P. bipes, small heterotrophic Gyrodinium spp., and naked ciliates (≤50 μm in cell length) sometimes have considerable potential grazing impact on the populations of E. gymnastica.     

Purification and Characterization of a Recombinant Bacteriophytochrome of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>

Phytochrome-like proteins have been recently identified in prokaryotes but their features and functions are not clear. We cloned a gene encoding the phytochrome-like protein (XoBphP) in a pathogenic bacteria, Xanthomonas oryzae pv. oryzae (Xoo) and investigated characteristics of the protein using a recombinant XoBphP. The N-terminal region of XoBphP containing the PAS/GAF/PHY domains is highly similar to most bacteriophytochromes, but Cys4, corresponding to Cys24 of DrBphP, isn’t involved in chromophore attachment. Recombinant XoBphP could bind a bilin molecule and a differential spectrum from Pr/Pfr shows that XoBphP has similar characteristics of known bacteriophytochromes with shifted absorption maxima of 683 and 757 nm for the Pr and Pfr forms. Unlike other bacteriophytochromes, XoBphP has no histidine kinase domain at C-terminus. The domain was predicted from amino-acid 279 to 342 with less significance than the required threshold. This result suggests that XoBphP probably has different signal transduction mechanisms for its intracellular function.     

Anti-angiogenic effects of the water extract of HangAmDan (WEHAD), a Korean traditional medicine

We investigated the anti-angiogenic effects of the water extract of HangAmDan (WEHAD), which is a crude extract of nine Korean medicinal substances of animal and plant origin. In human umbilical vein endothelial cells, WEHAD significantly inhibited bFGF-induced proliferation, adhesion, migration, and capillary tube formation. We used an antibody array to perform an analysis of signaling proteins, which showed up-regulated expression of various proteins including RAD51, RAD52, and p73, and down-regulated expression of pFAK. Blood vessel formation in a chick chorioallantoic membrane (CAM) treated with WEHAD was markedly reduced in length compared with a PBS-treated control group. These results suggest that inhibition of angiogenesis by WEHAD may be the mechanism of action for the anti-cancer effects of HAD.     

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.     

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.     

Effects of novel chalcone derivatives on α-glucosidase, dipeptidyl peptidase-4, and adipocyte differentiation in vitro

Chana series are new chalcone derivatives. To evaluate the possibility of Chana series as therapeutic agents of type 2 diabetes, the inhibitory effects of Chana series on the activities of α-glucosidase and DPP-4 were investigated using in vitro enzyme assays, and their effects on adipocyte differentiation were investigated in C3H10T1/2 cells. Chana 1 and Chana 7 among the Chana series showed significant inhibition of α-glucosidase activity. In DPP-4 enzyme assay, Chana 1 exhibited the highest inhibitory activity while Chana 7 did not. In MTT assay, Chana 1 did not show significant cytotoxicity up to a concentration of 250 μM, whereas cytotoxicity was observed with Chana 7 at a concentration of 300 μM. In addition, Chana 1 induced adipocyte differentiation. Therefore, Chana 1 showed inhibitory effects on α-glucosidase and DPP-4 as well as a stimulatory effect on adipocyte differentiation, suggesting that Chana 1 may be a potential beneficial agent for the treatment of type 2 diabetes.