In vitro efficacy of Linezolid on clinical strains of Mycobacterium tuberculosis and other mycobacteria

Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.     

Defective intracellular trafficking of uromodulin mutant isoforms

Medullary cystic kidney disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) are autosomal dominant renal disorders characterized by tubulo-interstitial fibrosis, hyperuricemia and medullary cysts. They are caused by mutations in the gene encoding uromodulin, the most abundant protein in urine. Uromodulin (or Tamm-Horsfall protein) is a glycoprotein that is exclusively expressed by epithelial tubular cells of the thick ascending limb of Henle's loop and distal convoluted tubule. To date, 37 different uromodulin mutations have been described in patients with MCKD/FJHN. Interestingly, 60% of them involve one of the 48 conserved cysteine residues. We have previously shown that cysteine-affecting mutations could lead to partial endoplasmic reticulum (ER) retention. In this study, as a further step in understanding uromodulin biology in health and disease, we provide the first extensive study of intracellular trafficking and subcellular localization of wild-type and mutant uromodulin isoforms. We analyzed a set of 12 different uromodulin mutations that were representative of the different kind of mutations identified so far by different experimental approaches (immunofluorescence, electron microscopy, biochemistry and in vivo imaging) in transiently transfected HEK293 and Madin-Darby canine kidney cells. We assessed protein processing in the secretory pathway and could demonstrate that although to different extent, all uromodulin mutations lead to defective ER to Golgi protein transport, suggesting a common pathogenetic mechanism in MCKD/FJHN.     

The assessment of DNA from marine organisms via a modified salting-out protocol

We developed a rapid, practical and non-toxic salting-out method for the extraction of DNA from marine organisms, and tested it on two representative species of Porifera and Cnidaria, both living in association with symbiotic zooxanthellae. We tested the efficiency of the protocol by comparing the output of the method for fresh tissue, frozen tissue and tissue stored in ethanol. It proved to be effective for extracting DNA in the case of the methods of preservation considered here, and for obtaining quantities of DNA comparable to those obtained via the traditional approach. The DNA from both species was of good quality. The DNA obtained was amplified by PCR using specific primers for the large ribosomal subunit, allowing the identification of the presence of both the host and symbiont genomes.     

13- and 14-membered macrocyclic ligands containing methylcarboxylate or methylphosphonate pendant arms: chemical and biological evaluation of their (153)Sm and (166)Ho complexes as potential agents for therapy or bone pain palliation

The stability constants of La(3+), Sm(3+) and Ho(3+) complexes with 13- and 14-membered macrocycles having methylcarboxylate (trita and teta) or methylphosphonate (tritp and tetp) arms were determined. All the ligands were labelled with (153)Sm and (166)Ho in order to evaluate the effect of the macrocyclic cavity size and type of appended arms on their in vitro and in vivo behaviour. The radiolabelling efficiency was found to be higher than 98% for all the complexes, except for those of tetp. All radiocomplexes studied are hydrophilic with an overall negative charge and low plasmatic protein binding. Good in vitro stability in physiological media and human serum was found for all complexes, except the (153)Sm/(166)Ho-teta, which are unstable in phosphate buffer (pH 7.4). In vitro hydroxyapatite (HA) adsorption studies indicated that (153)Sm/(166)Ho-tritp complexes bind to HA having the (166)Ho complex the highest degree of adsorption (>80%, 10 mg). Biodistribution studies in mice demonstrated that (153)Sm/(166)Ho-trita complexes have a fast tissue clearance with more than 95% of the injected activity excreted after 2 h, value that is comparable to the corresponding dota complexes. In contrast, the (153)Sm-teta complex has a significantly lower total excretion. (153)Sm/(166)Ho-tritp complexes are retained by the bone, particularly (166)Ho-tritp that has 5-6% (% I.D./g) bone uptake and also a high rate of total excretion. Thus, these studies support the potential interest of (153)Sm/(166)Ho-trita complexes for therapy when conjugated to a biomolecule and the potential usefulness of the (166)Ho-tritp complex in bone pain palliation.     

Increased substance P content in nerve fibers associated with mesenteric veins from deoxycorticosterone acetate (DOCA)-salt hypertensive rats

This study examined sensory nerves associated with mesenteric arteries and veins in sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Reactivity of arteries and veins to substances released from sensory nerves was also studied in vitro using computer-assisted video microscopy. Co-localization of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity (ir) was used to evaluate perivascular sensory nerves. Radioimmunoassay was used to quantify SP- and CGRP-ir content. Immunohistochemical studies revealed a plexus of SP/CGRP-ir nerves associated with arteries and veins. The intensity of SP-ir, but not CGRP-ir labeling was greater in arteries and veins from DOCA-salt compared to sham rats. RIA measurements revealed that the CGRP-ir content of arteries and veins was higher than the SP-ir content but there was a significant increase in SP-ir, but not CGRP-ir, content in arteries and veins from DOCA-salt rats. SP (0.03-1 microM) contracted veins and the NK-3 receptor agonist, senktide, mimicked this effect. There were no differences in SP or senktide reactivity of veins from sham or DOCA-salt rats. SP, but not senktide, relaxed KCl (40 mM) preconstricted arteries. CGRP (0.3 microM), acetylcholine (10 microM) and capsaicin (1 microM) relaxed KCl-preconstricted arteries and veins. The NK-1 receptor agonist, substance P methyl ester relaxed arteries but not veins. These data indicate that DOCA-salt hypertension is associated with upregulation of SP content in perivascular nerves. NK-3 receptors mediate venoconstriction which is unchanged in DOCA-salt hypertension. Increased release of SP from perivenous nerves might contribute to the increased venomotor tone in DOCA-salt hypertension.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Members only: induced systemic resistance to herbivory in a clonal plant network

The stoloniferous herb Trifolium repens was used to study the expression of induced systemic resistance (ISR) to the generalist caterpillar Spodoptera exigua in interconnected ramets of clonal fragments. The ISR was assessed as caterpillar preference in dual choice tests between control and systemically induced plants. The ISR was detected in young ramets, after inducing older sibling ramets on the same stolon by a controlled herbivore attack. However, older ramets did not receive a defense induction signal from younger ramets unless the predominant phloem flow was reversed by means of basal shading. This provides evidence for the notion that in T. repens the clone-internal expression of ISR is coupled to phloem transport and follows source–sink gradients. The inducibility of the genotypes was not linked to their constitutive ability to produce cyanide, implying the absence of a trade-off between these two defense traits. To our knowledge, this is the first study that explores ISR to herbivory in the context of physiological integration in potentially extensive clonal plant networks.