Role of beta(1)-integrins and their associated tetraspanin molecules in fibronectin-enhanced megakaryopoiesis

BACKGROUND: We have shown previously that fibronectin (FN) together with megakaryocyte (Mk) growth and development factor (MGDF) enhanced generation of Mk progenitors determined by colony-forming unit (CFU)-Mk assay. MGDF can activate beta(1)-integrins on progenitor cells and increase binding to FN. We studied the role of beta(1)-integrin-tetraspanin complexes by which FN may enhance megakaryopoiesis. METHODS: Cord blood CD34(+) cells were cultured for up to 8 days in serum-free medium containing IL-3, IL-6 and SCF with or without MGDF in the presence or absence of FN. Immunofluorescence flow cytometry was used to monitor changes in beta(1)-integrin-tetraspanin complexes. CFU-Mk assay was used to assess Mk commitment. RESULTS: The cocktail of cytokines irrespective of the presence of MGDF altered the percentage expression of beta(1)-integrins CD49d and CD49e on CD34(+) cells. CD49d expression fell initially (98% to 15%) and then rose to 75%, whereas CD49e progressively increased over the 8 days of culture, from 5.4% to 22%. However, with the addition of FN, similar changes in the expression of beta(1)-integrins were observed but the expression was maintained at a higher level. MGDF and FN increased expression of tetraspanin molecules, CD63 and CD151, as well as their co-expression with the beta(1)-integrins on both the CD34(+) and CD34(-) cells (e.g. and increase from 0% to 80% co-expression of CD49d and CD151 on CD34(+)). Blocking of beta(1)-integrins or the tetraspanin CD151 with the respective MAb reduced Mk progenitor generation in a stromal cell model. DISCUSSION: FN enhanced Mk progenitor generation through modulation of beta(1)-integrin-tetraspanin complexes, such as CD151/CD49d.     

Animal studies in endometriosis: a review

Endometriosis is a common women's health problem that is characterized by the presence of tissue resembling endometrium outside the uterus. The condition causes painful periods, chronic pelvic pain, and subfertility, which are potentially debilitating; and it affects millions of women worldwide. The diagnosis is made on visual inspection of the pelvis, usually at laparoscopy. The natural history is unknown, and well-controlled experiments are difficult to perform because of the need for repeated surgical procedures to assess endometriotic lesions over time. Thus, despite over 50 years' research, the cause of endometriosis remains unclear, and treatment options are limited. Animal models provide an invaluable tool to study risk factors, prevalence, and the natural history of endometriosis especially in those menstruating nonhuman primates that develop the disease spontaneously. Many of the practical problems associated with studying the disease in humans can therefore be overcome. The pathophysiology of endometriosis can also be investigated and new treatments assessed in both nonprimates and nonhuman primates, with "disease" induced by placing autologous uterine tissue in ectopic sites, or human endometrium in the case of nude mice. However, although nonprimates have obvious advantages as a model, the extent to which the induced lesions are truly representative of the disease itself is debatable. This review explores the value of the experimental models that have been used to date.     

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.     

Synergy of L-arginine and GHRP-2 stimulation of growth hormone in men and women: modulation by exercise

We investigated the ability of exercise, a multipathway, potent, physiological stimulus for GH release, to alter the synergistic interaction of L-arginine (A) and GH-related peptide (GHRP)-2 (G) observed at rest and the ability of gender to further modulate this putative interaction. Subjects (9 men and 9 early follicular phase women) completed 30 min of constant load aerobic exercise in combination with intravenous infusions of saline (S), A (30 g over 30 min), G (1 microg/kg bolus), or both (AG) in separate study sessions in randomly assigned order. Measures of GH release were logarithmically transformed for statistical analysis. Similar to rest, exercise maintained the rank order (AG > G > A > S) of effective stimulation of GH release for the key response measures in men or women, a gender disparity in the time to reach the maximal serum GH concentration, the calculated endogenous GH half-life, and the observed effect of preinfusion (basal) serum GH concentrations on determining secretagogue responsiveness. Exercise potentiated the individual stimulatory actions of A and G, while blunting the relative magnitude of the synergistic (supra-additive) interaction observed at rest. We infer from the present data that 1) exercise is likely to induce release of both GHRH and somatostatin, 2) L-arginine may facilitate the effect of exercise by limiting somatostatin release, 3) GHRP-2 could further enhance the stimulatory impact of exercise by opposing central actions of somatostatin and/or heightening endogenous GHRH release, and 4) gender strongly controls the relative but not absolute magnitude of A/G synergy both at rest and after exercise.     

OCT4 as a target of miR-34a stimulates p63 but inhibits p53 to promote human cell transformation

Human cell transformation is a key step for oncogenic development, which involves multiple pathways; however, the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC), we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung, breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and, OCT4, in turn, stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover, we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a, promotes p63 but suppresses p53 expression, which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and, most likely, also to the iPSC process.     

Plant size preference of Agapeta zoegana L. (Lepidoptera: Tortricidae), a root-feeding biological control agent of spotted knapweed

Agapeta zoegana L. (Lepidoptera: Tortricidae) is an oligophagous herbivore that was introduced to North America as a biological control agent of spotted knapweed, Centaurea stoebe L. subsp. micranthos (Gugler) Hayek (often called Centaurea maculosa Lam.). Spotted knapweed is a perennial plant that usually increases in size each year. A previous field study reported that more larvae were found on larger plants and that infested plants tended to be larger than uninfested ones. Precisely quantifying the size-specific attack rate can help us model the impact of this agent on the weed population and better understand the interspecific interactions to improve the effectiveness of biological control. Field data were analyzed to determine the relative preference of attack for each size class of the host plant. Plants were classified based on root diameter at 2 cm below the root crown. Although small plants (<3 mm root diameter) were more abundant in the field population, the highest infestation rates occurred in large plants. Chesson’s electivity index was generally positive for root diameters >3.5 mm, indicating preferential attack of large plants. Because of its host-size preference, A. zoegana is expected to primarily affect large plants, which is contrary to previous expectations. Quantifying the insect’s direct impact is a difficult challenge, which may require several field seasons of measuring accumulated damage on individual plants. In order to complement the biological control agents already established, foreign exploration should focus on finding an agent that attacks young knapweed plants. These results also indicate that the efficiency of sampling roots in the field to detect the presence of A. zoegana can be improved by choosing only the largest plants.     

Bortezomib enhances cytotoxicity of ex vivo-expanded gamma delta T cells against acute myeloid leukemia and T-cell acute lymphoblastic leukemia

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3⁺/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.     

Revised methods for the mass-rearing of the spotted knapweed biological control agent,Cyphocleonus achates (Coleoptera: Curculionidae), in field corrals

Cyphocleonus achates (Fahraeus), a root-feeding weevil introduced from Eurasia, is an effective biological control agent against spotted knapweed, Centaurea stoebe L. ssp. micranthos. Because C. achates is univoltine and does not fly, distribution of the weevil has been slow. To hasten the weevil's distribution, a rearing effort using field corrals was initiated at a facility in Corvallis, Montana. Procedures for mass-rearing the weevil in field corrals are described, with an emphasis on improvements over earlier methods. The described field-corral approach is effective and appropriate for producing C. achates for distribution in the western United States.     

iPSC-derived myelinoids to study myelin biology of humans

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