Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

Localization of motoneurons innervating the stylopharyngeus muscle in the cat

Recent investigations of the nucleus ambiguus (NA) have attempted to identify motoneurons associated with muscles of the larynx and pharynx. However, relatively little attention has been directed to the stylopharyngeus muscle, which is important in elevation of the pharynx in swallowing and speech. The present study was designed to identify the specific location of stylopharyngeus motoneurons within the brainstem. Horseradish peroxidase or fluorescent dye was injected into the stylopharyngeus muscle of 12 cats. Retrogradely labeled cells were located ipsilateral in the rostral NA and retrofacial nucleus. This is the first report to definitively localize stylopharyngeus motoneurons.     

Identification of four novel melanocortin 1 receptor (MC1R) gene variants in a Mediterranean population

The melanocortin 1 receptor (MC1R) gene is a major determinant of human pigmentation and specific allelic variants have been associated with red hair and sun sensitive skin types as well as increased skin cancer risk in Caucasian individuals. We screened for allelic variants the entire MC1R coding region of 100 unrelated individuals sampled from an Italian population who has darker pigmentary traits than populations analyzed to date. Twenty MC1R variants were identified, eighteen located at non-synonymous sites and two at synonymous sites. We report four novel MC1R allelic variants: C35Y (g.104G>A), V38M (g.112G>A), L44V (g.130C>G) and I120T (g.359T>C).     

Role of nitric oxide in the biology, physiology and pathophysiology of reproduction

Following its benchmark discovery, nitric oxide (NO) is nowknown to play important functional roles in a variety of physiologicalsystems. Within the vasculature, NO induces vasodilation, inhibitsplatelet aggregation, prevents neutrophil/platelet adhesionto endothelial cells, inhibits smooth muscle cell proliferationand migration, regulates programmed cell death (apoptosis) andmaintains endothelial cell barrier function. NO generated byneurons acts as a neurotransmitter, whereas NO generated bymacrophages in response to invading microbes acts as an antimicrobialagent. Because neurons, blood vessels and cells of the immunesystem are integral parts of the reproductive organs, and inview of the important functional role that NO plays in thosesystems, it is likely that NO is an important regulator of thebiology and physiology of the reproductive system. Indeed, inthe past 10 years, NO has established itself as a polyvalentmolecule which plays a decisive role in regulating multiplefunctions within the female as well as the male reproductivesystem. This review provides an overview of the role of NO invarious reproductive organs under physiological and pathologicalconditions.     

Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells

Regulation of intracellular pH (pH_i) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pH_i was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pH_i after induction of an acid load by removal of NH₄Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive²²Na⁺-uptake into Na⁺-loaded cells. Both results suggest the presence of a Na⁺/H⁺ antiport.(2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO ₃ ^– -Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl^–/HCO ₃ ^– exchange mechanism.(3) We found no evidence for a Na⁺/HCO ₃ ^– -cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pH_i in RPE: A Na⁺/H⁺ antiport responsible for recovery of pH_i from acid load, and a DIDS-sensitive Cl^–/HCO ₃ ^– exchange mechanism responsible for recovery of pH_i after alkalinization.Parts of this work jhave been published in abstract from [20, 21]