Ontogenetic profile of glutamate uptake in brain structures slices from rats: sensitivity to guanosine

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.     

Inherited lysozyme deficiency in rabbits. The absence of a primary isozyme of lysozyme as the cause of the condition

Lysozyme from normal and genetically lysozyme-deficient rabbits was extracted from three types of tissue: leukocytic (bone marrow), lymphoepithelial (thymus and appendix), and gastrointestinal (colon). Extracts were analyzed by electrophoretic, chromatographic, and kinetic techniques. Identification of at least two isozymes of rabbit lysozyme was made with these techniques. The distribution of the isozymes was tissue specific. Leukocytic and gastrointestinal isozymes were clearly distinguished, and a possible lymphoepithelial isozyme that resembled the gastrointestinal isozyme electrophoretically and chromatographically but not kinetically was identified. Mutant, lysozyme-deficient rabbits lacked completely a detectable leukocytic isozyme but had gastrointestinal and lymphoepithelial isozyme(s) indistinguishable from those of normal rabbits. By electrophoretic methods, the mutant rabbits were demonstrated to lack a protein band corresponding to that of the leukocytic isozyme of lysozyme from normal rabbits. These observations considered collectively were interpreted as evidence that at least two primary isozymes of lysozyme are present in rabbits and that inherited lysozyme deficiency in rabbits is caused by the absence of a single primary isozyme.     

Of sleep state and postnatal age on arousal responses induced by mild hypoxia in infants

STUDY OBJECTIVES: It has been suggested that mild hypoxia may not be a potent stimulus for arousal during sleep in infants because infants frequently fail to arouse from quiet sleep (QS). Our aim was to characterize arousal responses of sleeping infants in both active sleep (AS) and QS under normoxic and mildly hypoxic (15% O2) conditions over the first 6 months of life. PARTICIPANTS: Five healthy term and 6 healthy preterm infants were each studied at 2 to 5 weeks, 2 to 3 months, and 5 to 6 months postterm. All infants underwent daytime polysomnography during which nasal airflow was monitored using a purpose-built pneumotachograph. All infants were studied under both normoxic (21% O2) and hypoxic (15% O2, balance N2) conditions (presentation order randomized) in each sleep state at each study age. Tests were terminated at arousal, O2 saturation falling below 85%, or 5 minutes (failure to arouse). MEASUREMENTS: Probability of failure to arouse and mean arousal latency were compared between each experimental condition, with each infant serving as its own control. RESULTS: Infants aroused more frequently under hypoxic conditions than under normoxic conditions. Overall, arousal latencies were shorter during hypoxia compared to normoxia in both sleep states at each age. Arousal latencies were longer in QS compared to AS in both hypoxic and normoxic conditions. CONCLUSION: In sleeping infants, mild hypoxia serves as a stimulus for arousal in both AS and QS. Of particular significance is our finding that arousal from AS is readily elicited by mild hypoxia.     

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.     

Inhibins are the major activin ligands expressed during early thymocyte development.

Activins are members of the transforming growth factor-beta (TGFbeta) superfamily, which regulate cell differentiation processes. Here we report the first quantitative analysis of the expression of Activin/Inhibin ligands, type I and II receptors, as well as Smad proteins in fetal (E14-E16) and adult thymic subpopulations. Our data showed that Alk4, ActRIIA, ActRIIB, and Smads 2, 3, and 4, are expressed in fetal thymus (E14 > E15 > E16) and in thymocytes from adult mice (mostly in the double negative [DN] subpopulation). Ligand expression analysis showed that betaA, betaB, and alpha subunits were mainly detected in thymic stromal cells. Interestingly, alpha subunits were expressed at much higher levels compared to betaA and betaB subunits, demonstrating for the first time the potential role of Inhibins as important mediators during early T cell development. Our data indicate that Activin/Inhibin signaling could regulate the process of thymus organogenesis and early thymocyte differentiation, as it has been demonstrated for other members of the TGF-beta superfamily.     

X-ray diffraction computed tomography

Coherent scattering of x-ray photons leads to the phenomenon of x-ray diffraction, which is widely used for determining atomic structure in materials science. A technique [x-ray diffraction computed tomography (CT)] is described, analogous to conventional CT, in which the x-ray diffraction properties of a stack of two-dimensional object sections may be imaged. The technique has been investigated using a first generation (single pencil beam) CT scanner to measure small angle coherent scatter, in addition to the customary transmitted radiation. Diffraction data from a standard CT performance phantom obtained with this new technique and with an x-ray diffractometer are compared. The agreement is satisfactory bearing in mind the poor momentum resolution of our apparatus. The dose and sensitivity of x-ray diffraction CT are compared with those of conventional transmission CT. Diffraction patterns of some biological tissues and plastics presented in a companion paper indicate the potential of x-ray diffraction CT for tissue discrimination and material characterization. Finally, possibilities for refinement of the technique by improving the momentum resolution are discussed.     

Life cycle of the moss,Physcomitrella patens,in culture

Summary A method employing tissue culture techniques for growth of mosses is described. This method allows for the completion of the sexual life cycle of the mossPhyscomitrella patens (Hedw.) BSG under controlled conditions in a two month period. The moss system is useful for class demonstration or for research in the areas of development, genetics and plant physiology.     

Diurnal rhythms of leptin and ghrelin in the systemic circulation and in the gastric mucosa are related to food intake in rats

We investigated diurnal changes in leptin and ghrelin levels in the stomach and in the systemic circulation and their relation to food intake rhythms in Wistar rats housed at 22 °C with a 12-h light/dark cycle and free access to food and water. Animals were sacrificed every 3 h over a 24-h period. Leptin and ghrelin levels in serum and in the gastric mucosa were analysed by immunoassay. Leptin mRNA levels were determined in the gastric mucosa by RT-PCR and in different adipose tissue depots (epididymal, retroperitoneal and mesenteric) by Northern blot. Ghrelin mRNA levels were determined by Northern blot. Gastric and serum leptin levels displayed similar diurnal rhythms, rising during the dark phase and decreasing gradually during the light phase. Leptin expression in the different adipose tissue depots correlated positively with circulating leptin levels (P<0.05), although there were some depot-associated differences. Leptin mRNA levels in the mesenteric depot correlated positively with food intake (P<0.05). In blood, ghrelin levels rose sharply just before the onset of the dark phase and dropped suddenly just after. In the stomach, ghrelin levels were high during the fasting period of light and low during the night, and correlated inversely with food intake, gastric contents and serum leptin levels (P<0.05). Leptin and ghrelin in the stomach and in the systemic circulation thus show diurnal variations that are influenced by food intake rhythms. The results agree with a role for ghrelin as a stimulant of meal initiation.     

Predictive factors of virologic success in HIV-1-infected children treated with lopinavir/ritonavir

Predictive factors of the virologic success of the use of lopinavir/ritonavir (LPV/r) in HIV-infected children are unknown, especially in children who have been pretreated with protease inhibitors (PIs). This longitudinal, single-center, observational study included 69 children (21 PI-naive and 48 PI-experienced) who had received LPV/r for at least 3 months. The mean (+/- SD) age was 10.3 +/- 4.8 years, and the mean baseline of CD4 percentage and HIV-1 RNA was 14.9% +/- 9.8% and 4.8 +/- 1.05 log10 copies/mL, respectively. The mean duration of follow-up was 16.5 +/- 8.3 months. At 6, 12, and 18 months, 52%, 57%, and 49% of all children, respectively, had a viral load less than 50 copies/mL. The risk of virologic failure, defined as 2 consecutive viral loads greater than 1000 copies/mL, was significantly higher when the children were previously treated with PIs and when the baseline LPV mutation score exceeded 3 mutations. In the pretreated children, the ratio of the plasma LPV maximal concentration to the baseline LPV score mutation was also associated with failure, independently of resistance score. Finally, in children failing an LPV-containing regimen, accumulation of additional PI-associated resistance mutations was evidenced in viral isolates from children with prior PI treatment, even with viral replication levels less than 10,000 copies/mL. In pretreated children, LPV plasma levels should be optimized in an attempt to achieve sufficient drug concentrations to overcome the resistance level.     

Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins

Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the initial steps in lymphatic development. In Vegfc-/- mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/- mice developed cutaneous lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc alleles are required for normal lymphatic development.