Antibodies in cold stressed mice recognize a surface protein in Toxoplasma gondii tachyzoites.

Physical or psychological stressors are known to have significant consequences for immune function and the outcome of disease in human and animal models. In mice, cold water stress (CWS) has been shown to delay control of acute infection and reactivation of latent infections. Increased levels of parasite-specific IgG and IgM antibodies are observed when CWS is applied in the chronic phase. The present study examined the effects of a physical stressor, CWS, on tachyzoites antigens of Toxoplasma gondii, with particular emphasis on a low molecular weight antigen, 5 kDa, which seems to be recognized by antibodies from mice subjected to CWS in the chronic phase. This antigen is not recognized by antibodies from infected mice not subjected to CWS. Sera obtained from stressed and infected (CWS + INF) mice subjected to CWS during the chronic phase (CWS + INF + CWS) were used to harvest anti-5-kDa antibodies for immunolocalization studies. Tachyzoite lysate preparations were electrophoretically separated and transferred to nitrocellulose membranes. Strips of nitrocellulose containing tachyzoite antigens in the 4-10-kDa range were used to select for anti-5-kDa antibodies. Harvested anti-5-kDa localized this antigen on the surface of tachyzoites. This antigen was not present in bradyzoite preparations. Treatment with phosphatidylinositol-specific phospholipase C showed this antigen was not anchored to the cell membrane through glycosyl-phosphatidylinositol. Strong antibody responses in stressed animals during the chronic phase are associated with parasite reactivation. The 5-kDa antigen constitutes a unique immunogenic component of T. gondii, with significant diagnostic potential for identifying reactivation of latent infections.

Lysosomal trafficking functions of mucolipin-1 in murine macrophages.

BACKGROUND: Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes. RESULTS: We show that mucolipin-1 localizes to lysosomal compartments in RAW264.7 mouse macrophages that show subcompartmental accumulations of endocytosed molecules. Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane. CONCLUSION: Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

Serotonergic and glutamatergic neurons at the ventral medullary surface of the human infant: Observations relevant to central chemosensitivity in early human life.

Central chemoreception is the mechanism by which the brain detects the level of carbon dioxide (CO(2)) in the arterial blood and alters breathing accordingly in order to maintain it within physiological levels. The ventral surface of the medulla oblongata (VMS) of animals has long been recognized as a site of chemosensitivity, culminating in the recent identification of chemosensitive serotonergic (5-HT) and glutamatergic (Glut) neurons in this region. In this study, we analyzed the distribution of 5-HT and Glut neurons and their receptors in the arcuate nucleus (Arc) at the VMS of the human infant, using single-and double-label immunohistochemistry with specific antibodies. We also examined the expression of astrocytes, as experimental evidence suggests that astrocytes mediate, at least in part, central chemosensitivity via 5-HT and/or Glut receptors. We identified a small number of 5-HT neurons (approximately 5% of Arc neurons), distributed over the entire extent of the VMS, a large number of Glut neurons (approximately 95% of Arc neurons) that localized almost exclusively to the medial Arc, and a large number of astrocytes distributed across the entire extent of the VMS. The Arc also contained 5-HT(1A), kainate (GluR5), and 5-HT(2A) receptors, which localized predominantly to 5-HT neurons, glutamate neurons and astrocytes, respectively. Astrocytes also expressed the vesicular glutamate transporter 2 and low levels of 5-HT(1A) and kainate (GluR5) receptors, indicating that astrocytes may store and release glutamate, possibly in response to stimulation by 5-HT and/or Glut. These observations suggest that important functional interactions exist between 5-HT, glutamate, and astrocytes in the Arc. They also support the idea that the Arc is homologous to chemosensitive zones at the VMS in experimental animals. These data are important towards delineating the role of the human Arc in modulation of homeostasis, and its dysfunction in brainstem-associated pathologies such as the sudden infant death syndrome.

Multiple serotonergic brainstem abnormalities in sudden infant death syndrome.

CONTEXT: The serotonergic (5-hydroxytryptamine [5-HT]) neurons in the medulla oblongata project extensively to autonomic and respiratory nuclei in the brainstem and spinal cord and help regulate homeostatic function. Previously, abnormalities in 5-HT receptor binding in the medullae of infants dying from sudden infant death syndrome (SIDS) were identified, suggesting that medullary 5-HT dysfunction may be responsible for a subset of SIDS cases. OBJECTIVE: To investigate cellular defects associated with altered 5-HT receptor binding in the 5-HT pathways of the medulla in SIDS cases. DESIGN, SETTING, AND PARTICIPANTS: Frozen medullae from infants dying from SIDS (cases) or from causes other than SIDS (controls) were obtained from the San Diego Medical Examiner's office between 1997 and 2005. Markers of 5-HT function were compared between SIDS cases and controls, adjusted for postconceptional age and postmortem interval. The number of samples available for each analysis ranged from 16 to 31 for SIDS cases and 6 to 10 for controls. An exploratory analysis of the correlation between markers and 6 recognized risk factors for SIDS was performed. MAIN OUTCOME MEASURES: 5-HT neuron count and density, 5-HT(1A) receptor binding density, and 5-HT transporter (5-HTT) binding density in the medullary 5-HT system; correlation between these markers and 6 recognized risk factors for SIDS. RESULTS: Compared with controls, SIDS cases had a significantly higher 5-HT neuron count (mean [SD], 148.04 [51.96] vs 72.56 [52.36] cells, respectively; P<.001) and 5-HT neuron density (P<.001), as well as a significantly lower density of 5-HT(1A) receptor binding sites (P

Serotonin transporter abnormality in the dorsal motor nucleus of the vagus in Rett syndrome: potential implications for clinical autonomic dysfunction.

Autonomic dysfunction is prevalent in girls with Rett syndrome, an X-chromosome-linked disorder of mental retardation resulting from mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). This gene plays a role in regulating neuronal activity-dependent gene expression, including brain-derived neurotrophic factor (BDNF), which is a potent serotonergic (5-HT) neuronal growth factor. We analyzed selected parameters of the 5-HT system of the medulla in autopsied patients with Rett syndrome because of the role of BDNF in 5-HT cell development and because 5-HT plays a key role in modulating autonomic control. 5-HT neurons were identified by immunostaining for tryptophan hydroxylase, the biosynthetic enzyme for 5-HT. We quantitated the number of 5-HT cells in the medulla at 2 standardized levels in 11 Rett and 7 control cases. There was no significant difference in 5-HT cell number between the groups. We analyzed binding to the serotonin transporter (SERT) using the radioligand [(125)I]-RTI-55 with tissue autoradiography in 7 Rett and 5 controls in 9 cardiorespiratory-related nuclei. In the dorsal motor nucleus of the vagus (DMX) (preganglionic parasympathetic outflow), SERT binding for the control cases decreased significantly over time (p = 0.049) but did not change in the Rett cases (p = 0.513). Adjusting for age, binding between the Rett and control cases differed significantly in this nucleus (p = 0.022). There was a marginally significant age versus diagnosis interaction (p = 0.06). Thus, altered 5-HT innervation and/or uptake in the DMX may contribute to abnormal 5-HT modulation of this major autonomic nucleus in patients with Rett syndrome. These data suggest hypotheses concerning 5-HT modulation of vagal function for testing in MeCP2 knockout mice to understand mechanisms underlying autonomic dysfunction in patients with Rett syndrome.

BRCA1 requirement for the fidelity of plasmid DNA double-strand break repair in cultured breast epithelial cells.

The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.