Analysis of Genetic Variation in the Bovine SLC11A1 Gene, Its Influence on the Expression of NRAMP1 and Potential Association With Resistance to Bovine Tuberculosis.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where host genetics is thought to contribute to susceptibility or resistance. One of the genes implicated is the SLC11A1 gene, that encodes for the natural resistance-associated macrophage protein 1 (NRAMP1). The aim of this study was to identify SLC11A1 polymorphisms and to investigate any resulting functional differences in NRAMP1 expression that might be correlated with resistance/susceptibility to M. bovis infection. Sequencing of the SLC11A1 gene in cDNA isolated from Brown Swiss, Holstein Friesian, and Sahiwal cattle identified five single nucleotide polymorphisms (SNPs) in the coding region, but only one of these (SNP4, c.1066C>G, rs109453173) was present in all three cattle breeds and therefore warranted further investigation. Additionally, variations of 10, 11, and 12 GT repeats were identified in a microsatellite (MS1) in the SLC11A1 3'UTR. Measurement of NRAMP1 expression in bovine macrophages by ELISA showed no differences between cells generated from the different breeds. Furthermore, variations in the length of the MS1 microsatellite did not impact on NRAMP1 protein expression as analyzed by luciferase reporter assay. However, further analysis of the ELISA data identified that the presence of the alternative G allele at SNP4 was associated with increased expression of NRAMP1 in bovine macrophages. Since NRAMP1 has been shown to influence the survival of intracellular pathogens such as M. bovis through the sequestering of iron, it is possible that cattle expressing the alternative G allele might have an increased resistance to bTB through increased NRAMP1 expression in their macrophages.

An Olfactory Cilia Pattern in the Mammalian Nose Ensures High Sensitivity to Odors.

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those in weakly stimulated regions. Surprisingly, reduction in neuronal excitability or ablation of the olfactory G protein in OSNs does not alter the cilia length pattern, indicating that neither spontaneous nor odor-evoked activity is required for its establishment. Furthermore, the pattern is evident at birth, maintained into adulthood, and restored following pharmacologically induced degeneration of the olfactory epithelium, suggesting that it is intrinsically programmed. Intriguingly, type III adenylyl cyclase (ACIII), a key protein in olfactory signal transduction and ubiquitous marker for primary cilia, exhibits location-dependent gene expression levels, and genetic ablation of ACIII dramatically alters the cilia pattern. These findings reveal an intrinsically programmed configuration in the nose to ensure high sensitivity to odors.

G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons.

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR-G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

Spontaneous and sensory-evoked activity in mouse olfactory sensory neurons with defined odorant receptors.

Sensory systems need to tease out stimulation-evoked activity against a noisy background. In the olfactory system, the odor response profile of an olfactory sensory neuron (OSN) is dependent on the type of odorant receptor it expresses. OSNs also exhibit spontaneous activity, which plays a role in establishing proper synaptic connections and may also increase the sensitivity of the cells. However, where the spontaneous activity originates and whether it informs sensory-evoked activity remain unclear. We addressed these questions by examining patch-clamp recordings of genetically labeled mouse OSNs with defined odorant receptors in intact olfactory epithelia. We show that OSNs expressing different odorant receptors had significantly different rates of basal activity. Additionally, OSNs expressing an inactive mutant I7 receptor completely lacked spontaneous activity, despite being able to fire action potentials in response to current injection. This finding strongly suggests that the spontaneous firing of an OSN originates from the spontaneous activation of its G protein-coupled odorant receptor. Moreover, OSNs expressing the same receptor displayed considerable variation in their spontaneous activity, and the variation was broadened upon odor stimulation. Interestingly, there is no significant correlation between the spontaneous and sensory-evoked activity in these neurons. This study reveals that the odorant receptor type determines the spontaneous firing rate of OSNs, but the basal activity does not correlate with the activity induced by near-saturated odor stimulation. The implications of these findings on olfactory information processing are discussed.

Crucial role of copper in detection of metal-coordinating odorants.

Odorant receptors (ORs) in olfactory sensory neurons (OSNs) mediate detection of volatile odorants. Divalent sulfur compounds, such as thiols and thioethers, are extremely potent odorants. We identify a mouse OR, MOR244-3, robustly responding to (methylthio)methanethiol (MeSCH(2)SH; MTMT) in heterologous cells. Found specifically in male mouse urine, strong-smelling MTMT [human threshold 100 parts per billion (ppb)] is a semiochemical that attracts female mice. Nonadjacent thiol and thioether groups in MTMT suggest involvement of a chelated metal complex in MOR244-3 activation. Metal ion involvement in thiol-OR interactions was previously proposed, but whether these ions change thiol-mediated OR activation remained unknown. We show that copper ion among all metal ions tested is required for robust activation of MOR244-3 toward ppb levels of MTMT, structurally related sulfur compounds, and other metal-coordinating odorants (e.g., strong-smelling trans-cyclooctene) among >125 compounds tested. Copper chelator (tetraethylenepentamine, TEPA) addition abolishes the response of MOR244-3 to MTMT. Histidine 105, located in the third transmembrane domain near the extracellular side, is proposed to serve as a copper-coordinating residue mediating interaction with the MTMT-copper complex. Electrophysiological recordings of the OSNs in the septal organ, abundantly expressing MOR244-3, revealed neurons responding to MTMT. Addition of copper ion and chelator TEPA respectively enhanced and reduced the response of some MTMT-responding neurons, demonstrating the physiological relevance of copper ion in olfaction. In a behavioral context, an olfactory discrimination assay showed that mice injected with TEPA failed to discriminate MTMT. This report establishes the role of metal ions in mammalian odor detection by ORs.

A form of synaptically induced metabotropic glutamate receptor-dependent long-term depression that does not require postsynaptic calcium.

The calcium control hypothesis posits that postsynaptic calcium increases are required to trigger synaptic plasticity, with large increases inducing LTP and small increases inducing LTD. In CA1 of the hippocampus, however, LTD induced by chemical activation of metabotropic glutamate receptors (agonist-LTD) is independent of increases in postsynaptic calcium. Here we tested whether LTD induced by pairing of presynaptic stimulation with postsynaptic depolarization (synaptic-LTD) is similarly calcium-independent. This protocol induced an NMDA-dependent LTP when paired at 0mV, which was converted to mGluR-dependent LTD when paired at -20mV. The LTD was not blocked by calcium chelation, blockers of L- or T-type voltage-dependent calcium channels, or hyperpolarization to -70mV. We conclude that synaptically induced mGluR-dependent LTD, like agonist induced mGluR LTD, does not require calcium influx for its induction.

Distinct mechanisms contribute to agonist and synaptically induced metabotropic glutamate receptor long-term depression.

Metabotropic glutamate receptor mediated long-term depression (mGlu receptor LTD) is evoked in hippocampal area CA1 chemically by the agonist 3,5-Dihydroxyphenylglycine (DHPG, agonist LTD) and synaptically by paired-pulse low frequency stimulation (PP-LFS, synaptic LTD). We tested the hypothesis that different expression mechanisms regulate mGlu receptor LTD in 15-21 day old rats through neurophysiologic recordings in CA1. Several findings, in fact, suggest that agonist and synaptic mGlu receptor LTD are expressed through different mechanisms. First, neither LTD occluded the other. Second, a low calcium solution enhanced agonist LTD but did not alter synaptic LTD. Third, application of the mGlu receptor antagonist LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) reversed agonist LTD expression, but did not alter synaptic LTD. Finally, an N-type, voltage-dependent calcium channel antagonist, ω-conotoxin GVIA (CTX), reduced agonist LTD expression by 35%, but did not alter synaptic LTD. CTX also blocked the increase in the paired-pulse ratio associated with agonist LTD. This study cautions against assuming that agonist and synaptic LTD are equivalent as several components underlying their expression appear to differ. Moreover, the data suggest that agonist LTD, but not synaptic LTD, has a presynaptic, N-channel mediated component.

A multicenter experience with the Talent endovascular graft for the treatment of abdominal aortic aneurysms.

OBJECTIVE: The Talent endovascular graft has been used in the treatment of abdominal aortic aneurysms (AAAs) in more than 13,000 patients worldwide. However, information regarding the results of its use has been limited. This report describes the experience with 368 patients with AAAs who underwent treatment at four medical centers as part of an investigator-sponsored investigational device exemption trial. METHODS: Patients with AAAs were enrolled at four sites during a 32-month period from January 1999 to July 2001. All patients underwent treatment for infrarenal AAA with the Talent endovascular graft. Repair was performed with transrenal stent fixation under epidural (362/368 patients; 98.3%), local (4/368 patients; 1.1%), or general (2/368 patients; 0.5%) anesthesia. The average diameters were: maximum aortic aneurysm, 6.2 +/- 1.2 cm; proximal aortic fixation site, 2.6 +/- 0.4 cm; and distal iliac fixation site, 1.4 +/- 0.6 cm. Bifurcated grafts were used in 276 of 366 patients (75%), aortouniiliac in 57 of 366 patients (16%), and tube aortoaortic in 33 of 366 patients (9%). Multiple comorbid medical conditions were present in all patients (average, 4.7 conditions/patient). The mean age was 75.8 years, and 85% of the patients were male. Follow-up period ranged from 2 to 33 months (mean, 7.3 months). RESULTS: Endovascular graft deployment was accomplished in 366 of 368 patients. In the 263 patients followed for at least 6 months after endovascular repair, AAA diameter decreased by 5 mm or more in 83 patients (32%); diameter remained unchanged (change < 5 mm) in 157 patients (60%) and increased by 5 mm or more in 23 patients (8.7%). Major morbidity occurred in 46 of 368 patients (12.5%), and minor morbidity occurred in 31 of 368 (8.4%). The 30-day mortality rate was 1.9%. Secondary procedures were performed in 32 patients (8.7%). Late rupture occurred in two patients, and late deaths unrelated to AAA occurred in 32 patients (8.7%) during the follow-up period. The primary technical success rate for all patients was 93.4%. The 30-day primary procedural success rate was 73.3%. The 30-day secondary procedural success rate was significantly higher at 85.8%. Computed tomographic scan was performed within 1 month after surgery in 349 patients. An endoleak was present in 43 of 349 patients (12.3%). These endoleaks were comprised of 10 attachment site (type I; 2.9%), 31 retrograde side-branch (type II; 8.9%), and two transgraft (type III; 0.6%). CONCLUSION: These midterm findings show a high degree of technical and procedural success achieved in a patient population with extensive comorbid medical illnesses with low perioperative morbidity and mortality rates. Further follow-up study will be necessary to determine the effectiveness of the Talent endograft for the long-term treatment of AAA.

An in situ adjustable endovascular graft for the treatment of abdominal aortic aneurysms.

Twenty-nine patients underwent placement of the Teramed Ariba Endovascular Graft System (Maple Grove, Minn) as part of a European Feasibility study (14 patients) and a US phase I trial (15 patients). Salient features of this modular endograft system include a crimped seamless polyester bifurcation graft supplied in three diameters and three iliac limb lengths, three types of nitinol stents including a suprarenal stent with aortic barbs, a flexible delivery system capable of controlled incremental sheath retraction, a flexible tapered lead balloon, and a telescoping technique for adjusting the length of graft coverage during surgery over a range of 3 cm. Twenty-eight of the 29 patients met the primary objective of this evaluation, which was to confirm the safety of the system, defined as the absence of major device-related adverse events and type I, III, or IV endoleaks within 1 month of implantation. Three major adverse events occurred within 1 month of discharge: renal failure, which was related to deployment of the device close to the renal arteries; pulmonary edema, which was related to the procedure but not the device; and peripheral ischemia, which was related to the patient's pre-existing condition. Seven patients had type II endoleaks noted by means of computed tomography scanning at 1 month; the endoleaks were identified by means of angiography and classified at the time of surgery. There were no deaths, aneurysm ruptures, stent-graft migrations, stent fractures, graft ruptures, graft thromboses, or surgical conversions at 1 month. This early clinical experience indicates that the Ariba Bifurcated Endovascular System can be safely implanted.