FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.     

Bone staining in waterlogged deposits: a preliminary contribution to the interpretation of near-shore find accumulation at the Schöningen 13II-4 ‘Spear-Horizon’ site,Lower Saxony,Germany

Abstract

The Schöningen 13II-4 ‘Spear Horizon’ site is famous for the excellent preservation of 300,000-year-old Palaeolithic hunting weapons, including nine wooden spears and a lance, deposited on the shores of a former interglacial lake in association with a large assemblage of well-preserved and butchered animal bones, mainly from horse. Some bones show distinct areas of dark staining, thought to be derived from contact with decaying plant remains along the shores of the lake. It was decided to test this theory and try to determine experimentally where bone staining was most likely to occur on the littoral zone. Modern horse and cow bones were fastened along parallel transects at two locations and the installations were left for several months. Black stains appeared on some bones in the shallows, but not on bones deposited on permanently dry land or in deeper water. Within the 10 m wide band of bones in the main concentration at the Schöningen site, there is a high incidence of bone staining, indicating accumulation of finds along a shallow lake margin. By using GIS, additional clusters of stained bones in the eastern part of the site were revealed and may indicate shorelines when water levels in the lake were lower.     

Expression and function of Xenopus laevis p75(NTR) suggest evolution of developmental regulatory mechanisms

Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75(NTR) has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75(NTR) imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75(NTR) function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75(NTR) genes from Xenopus, which we have termed p75(NTR)a and p75(NTR)b. We then cloned two different cDNAs, both of which encompass the full coding region of p75(NTR)a. Early in development both p75(NTR)a and p75(NTR)b are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75(NTR)a expression largely continues to parallel p75(NTR) expression in other species. However, Xenopus p75(NTR)a is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75(NTR) and each of two truncated mutants in developing retina reveal that p75(NTR) probably signals for cell survival in this system. This result contrasts with the reported role of p75(NTR) in developing retinae of other species, and the possible implications of this difference are discussed.     

Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors. Biochemical identification and characterization of new subunits.

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of alpha (39,000), beta (36,000), and gamma (approximately 7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct alpha and two distinct beta gamma activities in frog (Rana catesbeiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these alpha fractions was proportional to the amount of the M(r) 39,000 protein. The first alpha was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but alpha subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second alpha s were the same protein; however, the third alpha showed different characters as follows. We designated alpha in the first two fractions as alpha 1, and alpha in the third fraction as alpha 2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both alpha subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/mol of alpha. Compared with alpha 1, alpha 2 required larger amounts of rhodopsin and beta gamma for the binding of a hydrolysis-resistant GTP analog. alpha 2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both alpha 1 and alpha 2 complexed with beta gamma or beta delta (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two beta gamma fractions revealed that the main fraction was composed of a beta gamma complex; however, the second active fraction was composed of beta complexed with delta (M(r) 12,000). Compared with beta gamma, beta delta stimulated GTP binding to alpha 1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five beta and two gamma isoforms in beta gamma. Only one beta isoform was present in beta delta. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.     

Features of Child Food Insecurity after the 2010 Haiti Earthquake: Results from Longitudinal Random Survey of Households

Background

Recent commentary on the health consequences of natural disasters has suggested a dearth of research on understanding the antecedents prior to the disaster that are associated with health consequences after the disaster. Utilizing data from a two-wave panel survey of Port-au-Prince, Haiti, conducted just prior to and six weeks after the January 2010 earthquake, we test factors prior to the quake hypothesized to be associated with food insecurity after the quake.

Methods

Using random Global Positioning System (GPS) sampling, we re-interviewed 93.1% (N = 1732) of the original 1,800 households interviewed in 2009. Respondents were queried with regard to mortalities, injuries, food security, housing, and other factors after the quake.

Findings

Child food insecurity was found to be common on all three indices of food security (17.2%–22.6%). Additionally, only 36.5% of school-aged children were attending school prior to the quake. Findings suggest that prior schooling was associated with a substantial reduction on food insecurity indices (OR 0.62–0.75). Findings further suggest that several household characteristics were associated with food insecurity for children. Prior chronic/acute illnesses, poor living conditions, remittances from abroad, primary respondent mental health, and histories of criminal and other human rights violations committed against family members prior to the quake were associated with food insecurity after the earthquake. Earned household income after the quake was only associated with one of the measures of food insecurity.

Interpretation

Food insecurity for children was common after the quake. Those households vulnerable on multiple dimensions prior to the quake were also vulnerable to food insecurity after the quake. Remittances from abroad were leading protective factors for food security. Because Haiti is well known for the potentiality of both hurricanes and earthquakes, reconstruction and redevelopment should focus on ameliorating potential vulnerabilities to poor outcomes in these natural disasters.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.     

Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of alpha-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the alpha-amino group to glutamate. Measurements of synthesis of (14)C-glutamate and of (14)C-glutamine from H(14)CO(3) have shown that (14)C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The specific radioactivity of (14)C-glutamine indicated that approximately 30% of glutamine was derived from (14)CO(2) fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of (14)C-glutamate and (14)C-glutamine from H(14)CO(3)(-) was inhibited by 31%. These results suggest that transamination of alpha-ketoglutarate to glutamate in Müller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H(14)CO(3)(-). (14)C-metabolites in brain extracts were measured to determine net incorporation of (14)CO(2) and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from (14)CO(2) fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for approximately 60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.     

Time course of NADH oxidase, inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/WOR rat.

This study investigated the time course of NADH oxidase, a source of superoxide in the vascular endothelium, inducible nitric oxide synthase (iNOS), and peroxynitrite (ONOO(-)) in the BBZ/Wor rat, a spontaneous model of noninsulin dependent diabetes (NIDDM). Colloidal gold-labeled immunocytochemical studies of iNOS and nitrotyrosine, a marker for OONO(-), were done on sections of retinas from male BBZ/Wor rats in which NADH oxidase was localized by cerium derived cytochemistry at three time points: pre-diabetes (prior to the onset of hyperglycemia); new onset diabetes (2-6 days after onset of hyperglycemia); and chronic diabetes (4-18 months after onset of hyperglycemia). Control retinas were from age matched non-diabetic BB(DR)/Wor rats. The percentage of blood vessels positive for NADH oxidase increased significantly (P = 0.05) in new onset (64.2 +/- 6.5%) and chronic diabetes (83.2 +/- 11.4%), as compared to pre-diabetes (25.8 +/- 5.6%) and nondiabetic controls (33.6 +/- 15.9%). The percentage of blood vessels positive for iNOS immunoreactivity was significantly higher in new onset diabetic retinas (69.6 +/- 5.88%, P = 0.0001; 8.9 +/- 3.29 colloidal gold particles (cgp) /50 microm(2)) than in chronic diabetic retinas (49.9 +/- 9.75%; 7.9 +/- 5.12 cgp) and both were significantly higher (P = 0.0001) than in prediabetic (3.7 +/- 0.81%; 0.4 +/- 0.56 cgp) and nondiabetic control retinas (8.7 +/- 4.66%; 1.2 +/- 1.40 cgp). In new onset diabetes, levels of nitrotyrosine immunoreactivity (60.8 +/- 16.91 cgp) were significantly higher (P = 0.0001) than those in chronic diabetes (29.5 +/- 4.31 cgp); both were significantly higher (P = 0.0001) than those in prediabetic (8.2 +/- 1.70 cgp) and nondiabetic retinas (9.0 +/- 1.87 cgp). There was no cumulative increase in nitrotyrosine in the chronic diabetic retinas as a function of time. In rats with diabetes there was disruption of the inner blood-retinal barrier. These results suggest that iNOS and ONOO(-) may contribute to retinal damage in diabetes from the onset of hyperglycemia in NIDDM.