c-Rel is essential for the development of innate and T cell-induced colitis

Inflammatory bowel disease is a chronic inflammatory response of the gastrointestinal tract mediated in part by an aberrant response to intestinal microflora. Expression of IL-23 subunits p40 and p19 within cells of the innate immune system plays a central role in the development of lower bowel inflammation in response inflammatory challenge. The NF-kappaB subunit c-Rel can regulate expression of IL-12/23 subunits suggesting that it could have a critical role in mediating the development of chronic inflammation within the lower bowel. In this study, we have analyzed the role of c-Rel within the innate immune system in the development of lower bowel inflammation, in two well-studied models of murine colitis. We have found that the absence of c-Rel significantly impaired the ability of Helicobacter hepaticus to induce colitis upon infection of RAG-2-deficient mice, and ameliorated the ability of CD4(+)CD45RB(high) T cells to induce disease upon adoptive transfer into RAG-deficient mice. The absence of c-Rel interfered with the expression of IL-12/23 subunits both in cultured primary macrophages and within the colon. Thus, c-Rel plays a critical role in regulating the innate inflammatory response to microflora within the lower bowel, likely through its ability to modulate expression of IL-12/23 family members.     

Structural abnormalities in neurons are sufficient to explain the clinical disease and fatal outcome of experimental rabies in yellow fluorescent protein-expressing transgenic mice

Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.     

Overweight is associated with decreased cognitive functioning among school-age children and adolescents

Objective: Childhood overweight and obesity have increased substantially in the past two decades, raising concerns about their psychosocial and cognitive consequences. We examined the associations between academic performance (AP), cognitive functioning (CF), and increased BMI in a nationally representative sample of children. Methods and Procedures: Participants were 2,519 children aged 8–16 years, who completed a brief neuropsychological battery and measures of height and weight as a part of the Third National Health and Nutrition Examination Survey, a cross‐sectional survey conducted between 1988 and 1994. Z‐scores were calculated for each neuropsychological test, and poor performance was defined as z‐score <2. Results: The association between BMI and AP was not significant after adjusting for parental/familial characteristics. However, the associations between CF remained significant after adjusting for parental/familial characteristic, sports participation, physical activity, hours spent watching TV, psychosocial development, blood pressure, and serum lipid profile. Z‐scores on block design (a measure of visuospatial organization and general mental ability) among overweight children and children at risk of overweight were below those of normal‐weight children by 0.22 (s.e. = 0.16) and 0.10 (s.e. = 0.10) unit, respectively (P for trend <0.05). The odds of poor performance on block design were 1.97 (95% confidence interval: 1.01–3.83) and 2.80 (1.16–6.75), respectively, among children at risk or overweight compared to normal‐weight peers. Discussion: Increased body weight is independently associated with decreased visuospatial organization and general mental ability among children. Future research is needed to determine the nature, persistence, and functional significance of this association.     

Control of excitatory synaptic transmission by C-terminal Src kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.     

High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.     

Impact of duration versus frequency of probing by Homalodisca vitripennis (Hemiptera: Cicadellidae) on inoculation of Xylella fastidiosa

Successful infection of the plant pathogenic bacterium Xylella fastidiosa (Wells) from an infected plant to a new host involves three main steps: 1) acquisition of the bacterium by a vector; 2) inoculation of a noninfected host plant by the vector; and 3) establishment of sufficient titers of X. fastidiosa in the host plant to sustain a chronic infection. Understanding the basic biology of the transmission process is a key to limiting the spread of plant diseases induced by X. fasdidiosa and reducing agricultural losses, especially those experienced in California since the introduction of a new vector, Homalodisca vitripennis (Germar) (Hemiptera, Cicadellidae) (formerly H. coagulata Say), the glassy-winged sharpshooter. In this study, H. vitripennis adults that acquired X. fastidiosa were allowed access to chrysanthemum plant cuttings for 30, 60, 90, or 120 min. The numbers of X. fastidiosa acquired (i.e., cells present in the insect foregut) and the number inoculated to the plant cuttings were separately determined using quantitative real-time polymerase chain reaction (PCR). In addition, the number of times glassy-winged sharpshooter stylets probed plant cuttings and the amount of time glassy-winged sharpshooter spent actively ingesting were monitored using video surveillance. Linear regression did not indicate a relationship between the number of X. fastidiosa cells inoculated into the plant cutting and either the titer of pathogen present in the insect or amount of time spent ingesting per probe. However, the number of probes significantly influenced the number of X. fastidiosa cells inoculated. Due to the highly variable nature of transmission, our model could not account for all observed variation as indicated by low R2 values. However, our results suggest that the mechanism of transmission is dependent on probing behaviors more than ingestion duration.     

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.     

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.     

Characterization of porous poly(D,L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells

This study presents the application of the porous poly(D,L-lactic-co-glycolic acid) (PLGA) sponges fabricated from an organic solvent free supercritical gas foaming technique. Two formulations of PLGA sponges with different co-polymer compositions (85:15 and 50:50) were fabricated as novel scaffolds to guide human hepatoma cell line, Hep3B cell growth in vitro. The PLGA sponges showed desirable biodegradability and exhibited uniform pore size distribution with moderate interconnectivity. It was observed in this study that cells cultured on PLGA sponges showed lower proliferation rate as compared to the control during 14 days of culture as measured by using total DNA and methylthiazol tetrazolium (MTT) assays. However, the cells cultured on the sponges tended to aggregate to form cell islets which were able to express better hepatic functions. The enzyme-linked immunosorbent assay (ELISA) results showed that the cell-sponge constructs secreted 1.5-3.0 times more albumin than the control when normalized to cellular content. In a similar fashion, its detoxification ability was also predominantly higher than that of the control as indicated by the ethoxyresorufin-O-deethylase (EROD) results. By comparing the cells growing on the two formulations of PLGA sponges, it was found that the PLGA 85:15 sponge exhibited better conductive and desirable environment for hep3B cells as justified by better cell infiltration, higher proliferation and hepatic function than the PLGA 50:50 sponge.     

BDNF-TrkB signaling interacts with the GABAergic system to inhibit rhythmic swallowing in the rat

Brain-derived neurotrophic factor (BDNF) acts as an anorexigenic factor in the dorsal vagal complex (DVC) of the adult rat brain stem. The DVC contains the premotoneurons controlling swallowing, a motor component of feeding behavior. Although rats with transected midbrain do not seek out food, they are able to swallow and to ingest food. Because BDNF and tropomyosin-related kinase B (TrkB) receptors are expressed in the DVC, this study hypothesized that BDNF could modify the activity of premotoneurons involved in swallowing. Repetitive electrical stimulation of the superior laryngeal nerve (SLN) induces rhythmic swallowing that can be recorded with electromyographic electrodes inserted in sublingual muscles. We show that a microinjection of BDNF in the swallowing network induced a rapid, transient, and dose-dependant inhibition of rhythmic swallowing. This BDNF effect appeared to be mediated via TrkB activation, since it no longer occurred when TrkB receptors were antagonized by K-252a. Interestingly, swallowing was inhibited when subthreshold doses of BDNF and GABA were coinjected, suggesting a synergistic interaction between these two signaling substances. Moreover, BDNF no longer had an inhibitory effect on swallowing when coinjected with bicuculline, a GABA(A) receptor antagonist. This blockade of BDNF inhibitory effect on swallowing was reversible, since it reappeared when BDNF was injected 15 min after bicuculline. Finally, we show that stimulation of SLN induced a decrease in BDNF protein within the DVC. Together, our results strongly suggest that BDNF inhibits swallowing via modulation of the GABAergic signaling within the central pattern generator of swallowing.