Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.     

Magnesium intake and serum C-reactive protein levels in children.

The objective of this study was to determine whether magnesium consumption is associated with inflammation (C-reactive protein [CRP]) in children. The study was an analysis of child (age 6-17 years) participants in the cross-sectional, nationally representative National Health and Nutrition Examination Survey (NHANES). Children consuming less than 75% of RDA were 1.94 times more likely (p < 0.05) to have elevated serum CRP levels than children consuming above the RDA. In adjusted analyses controlling for demographics, cardiovascular risk factors, and BMI, children with consumption of less than 75% RDA were 58% more likely to have elevated CRP (OR 1.58, 95% CI 1.07-infinity). Children with intakes below the RDA are more likely to have elevated CRP levels.     

Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.     

Inhibitors of cytochrome P450 4A suppress angiogenic responses

Cytochrome P450 enzymes of the 4A family (CYP4A) convert arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) in blood vessels of several vascular beds. The present study examined the effects of inhibiting the formation of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016) on the mitogenic response of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs) in vitro, and on growth factor-induced angiogenesis in the cornea of rats in vivo. HET0016 (10 micromol/L and 20 microg, respectively) abolished the mitogenic response to VEGF in HUVECs and the angiogenic response to VEGF, basic fibroblast growth factor, and epidermal growth factor in vivo by 80 to 90% (P < 0.001). Dibromododecenyl methylsulfonimide (DDMS), a structurally and mechanistically different inhibitor of 20-HETE synthesis, also abolished angiogenic responses when tested with VEGF. Additionally, administration of the stable 20-HETE agonist, 20-hydroxyeicosa-6(Z) 15(Z)-dienoic acid (WIT003) induced mitogenesis in HUVECs and angiogenesis in the rat cornea in vivo. We studied the ability of HET0016 to alter the angiogenic response in the rat cornea to human glioblastoma cancer cells (U251). When administered locally into the cornea, HET0016 (20 microg) reduced the angiogenic response to U251 cancer cells by 70%. These results suggest that a product of CYP4A product, possibly 20-HETE, plays a critical role in the regulation of angiogenesis and may provide a useful target for reduction of pathological angiogenesis.     

Comparing the effectiveness of home visiting paraprofessionals and mental health professionals delivering a postpartum depression preventive intervention: a cluster-randomized non-inferiority clinical trial

To determine whether pregnant women receiving the Mothers and Babies group–based intervention exhibited greater depressive symptom reductions and fewer new cases of major depression than women receiving usual community-based services, and to examine whether groups run by paraprofessional home visitors and mental health professionals yielded similar depressive symptom reductions and prevention of major depression. Using a cluster-randomized design, 37 home visiting programs were randomized to usual home visiting, Mothers and Babies delivered via home visiting paraprofessionals, or Mothers and Babies delivered via mental health professionals. Baseline assessments were conducted prenatally with follow-up extending to 24 weeks postpartum. Eligibility criteria were ≥ 16 years old, ≤ 33 gestation upon referral, and Spanish/English speaking. Depressive symptoms at 24 weeks postpartum was the primary outcome. Eight hundred seventy-four women were enrolled. Neither intervention arm was superior to usual care in decreasing depressive symptoms across the sample (p = 0.401 home visiting paraprofessional vs. control; p = 0.430 mental health professional vs. control). Post hoc analyses suggest a positive intervention effect for women exhibiting mild depressive symptoms at baseline. We have evidence of non-inferiority, as the model-estimated mean difference in depressive symptoms between intervention arms (0.01 points, 95% CI: −0.79, 0.78) did not surpass our pre-specified margin of non-inferiority of two points. Although we did not find statistically significant differences between intervention and control arms, non-inferiority analyses found paraprofessional home visitors generated similar reductions in depressive symptoms as mental health professionals. Additionally, Mothers and Babies appears to reduce depressive symptoms among women with mild depressive symptoms when delivered by mental health professionals. This trial is registered on ClinicalTrials.gov (initial post: December 1, 2016; identifier: NCT02979444).

     

Calibration of the maize yg2 assay using gamma radiation and ethylmethanesulfonate

A standard protocol for the yellow-green-2 (yg2) forward mutation assay in Zea mays is proposed. A detailed calibration of the assay using 137Cs gamma rays and ethylmethanesulfonate (EMS) was conducted. Gamma ray-induced mutant sectors in leaves 4 and 5 exhibited one-hit kinetics. The radiation doses ranged from 25 to 500 rads. The mean induced mutation rate per rad of gamma radiation was 4.54 X 10(-6). This value was constant for the primordial cells of leaves 4 or 5. The induction of forward mutation by EMS also exhibited one-hit kinetics in the concentration range 0.25-20 mM (0.33-23.54 mumol EMS/kernel). The mean induced mutation rate per mM EMS was 1.79 X 10(-4), and the mean induced mutation rate per mumol of EMS per kernel was 1.52 X 10(-4). Using the induced mutation rates for gamma radiation and EMS, the rad equivalent was calculated. One rad of gamma radiation is equivalent to the exposure of a 2.53 X 10(-5) M EMS solution or to 2.99 X 10(-8) mol of EMS per kernel.     

Human gender differences in fibrinolytic responses to exercise training and their determinants

Endurance exercise training improves fibrinolysis, but this training-induced adaptation may differ somewhat between men and women. We sought to determine whether the potential gender differences in training-induced changes in selected fibrinolysis measures were related to changes in adiposity and/or plasma lipoprotein lipid levels. Seventeen men and 28 women, 50-75 years old, who were generally overweight to obese, were assessed for plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) activity, t-PA antigen and plasma lipoprotein-lipid levels, and body composition before and after 6 months of endurance exercise training while on a low-fat diet. At baseline, there were no differences in fibrinolytic measures between the men and women. Baseline levels of these fibrinolytic markers in both men and women were primarily related to other fibrinolytic measures and body composition, with a smaller contribution from plasma high-density lipoprotein cholesterol (HDL-C) levels. Exercise training reduced t-PA antigen levels in both men and women, but the reduction was significantly greater in men (-1.6 +/- 0.3 versus -0.5 +/- 0.2 ng ml(-1), P = 0.007). Exercise training decreased PAI-1 activity more in men than in women (-2.6 +/- 1.4 versus +0.9 +/- 0.9 IU ml(-1), P = 0.03). Men and women both showed increased t-PA activity with exercise training to the same extent (+0.38 +/- 0.12 versus +0.36 +/- 0.24 U ml(-1)). The changes in fibrinolytic measures with exercise training in men and women were correlated with changes in other fibrinolytic measures, although in men abdominal fat changes were a strong predictor of fibrinolytic changes with training. These findings suggest that training-induced improvements in endogenous fibrinolysis markers are somewhat greater in men compared to women and may be more strongly associated with abdominal obesity in men.     

Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions

CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules.     

An unusual surface peroxiredoxin protects invasive Entamoeba histolytica from oxidant attack

Peroxiredoxins are an important class of antioxidant enzymes found from Archaea to humans, which reduce and thereby detoxify peroxides and peroxynitrites. The major thiol-containing surface antigen of the invasive ameba, Entamoeba histolytica, is a peroxiredoxin and is likely to be important during the transition from the anaerobic environment of the large intestine to human tissues. The closely related species, Entamoeba dispar, is incapable of invasion and more sensitive to hydrogen peroxide, yet also has a peroxiredoxin. We cloned and expressed the two active recombinant enzymes and found that their activity was similar by a fluorometric stopped-flow assay, giving a Km of <10 microM for hydrogen peroxide. Three monoclonal antibodies produced to recombinant E. histolytica peroxiredoxin cross-reacted with Entamoeba dispar.E. histolytica contains as much as 50 times more peroxiredoxin than E. dispar as demonstrated by a sensitive capture ELISA. In addition, the peroxiredoxin is present largely on the outer surface of the cell, in contrast to E. dispar. This unusual peroxiredoxin localizes to the site of parasite-host cell contact where it can effectively counteract oxidants generated by host cells, thus facilitating invasion.     

A developmental look at an auditory illusion: The precedence effect

Infants aged 2 and 6 months were tested with the precedence effect, an auditory phenomenon involving sound localization. Each infant was tested with two types of stimuli: sound from a single loudspeaker and precedence-effect sounds produced by the same sound put through two loudspeakers, with one output leading the other by 7 msec. Older infants localized precedence-effect stimuli as they did single-source stimuli, indicating that they perceived this phenomenon as expected. Two-month-olds turned their heads toward single-source sounds, but did not localize precedence-effect sounds, suggesting that that more difficult perceptual task had not been achieved at this age. In general, headturning toward sound proved far more difficult to elicit in younger infants. A click train was ineffective, but a tape-recorded human voice elicited above-chance low-level turning. The developmental changes in auditory behavior are discussed in terms of the rapid growth of the auditory cortex.