A Missense Mutation in PRPF6 Causes Impairment of pre-mRNA Splicing and Autosomal-Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration that leads to progressive visual-field constriction and blindness. Although the disease manifests only in the retina, mutations in ubiquitously expressed genes associated with the tri-snRNP complex of the spliceosome have been identified in patients with dominantly inherited RP. We screened for mutations in PRPF6 (NM_012469.3), a gene on chromosome 20q13.33 encoding an essential protein for tri-snRNP assembly and stability, in 188 unrelated patients with autosomal-dominant RP and identified a missense mutation, c.2185C>T (p.Arg729Trp). This change affected a residue that is conserved from humans to yeast and cosegregated with the disease in the family in which it was identified. Lymphoblasts derived from patients with this mutation showed abnormal localization of endogenous PRPF6 within the nucleus. Specifically, this protein accumulated in the Cajal bodies, indicating a possible impairment in the tri-snRNP assembly or recycling. Expression of GFP-tagged PRPF6 in HeLa cells showed that this phenomenon depended exclusively on the mutated form of the protein. Furthermore, analysis of endogenous transcripts in cells from patients revealed intron retention for pre-mRNA bearing specific splicing signals, according to the same pattern displayed by lymphoblasts with mutations in other PRPF genes. Our results identify PRPF6 as the sixth gene involved in pre-mRNA splicing and dominant RP, corroborating the hypothesis that deficiencies in the spliceosome play an important role in the molecular pathology of this disease.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.

Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis.

Hajdu-Cheney syndrome is a rare autosomal dominant skeletal disorder with facial anomalies, osteoporosis and acro-osteolysis. We sequenced the exomes of six unrelated individuals with this syndrome and identified heterozygous nonsense and frameshift mutations in NOTCH2 in five of them. All mutations cluster to the last coding exon of the gene, suggesting that the mutant mRNA products escape nonsense-mediated decay and that the resulting truncated NOTCH2 proteins act in a gain-of-function manner.

Functional significance of first metatarso-phalangeal joint arthrodesis : FM101

Introduction: Indications for arthrodesis of the first metatarsophalangeal joint (MTP1) are commonly arthrosis (hallux rigidus), rheumatoid arthritis, failed hallux valgus surgery, severe hallux valgus, infectious arthritis, fractures and neuroarthropathies. Many reports focus on technical and radiological issues but few studies emphasize the functional outcome considering daily activities, sports and expectation of the patient. Method: We retrospectively reviewed the patients who underwent MTP1-arthrodesis from 2002 to 2005 in our institution. Clinical and radiological results were assessed but we specially focussed on the functional outcome. Scoring systems used were the SF-12, EQ-5D, PASI, FFI and AOFAS (10 points given to MTP1 mobility) scales. Results: 61 of 64 consecutive patients were evaluated. Female to male ratio was 49:15, mean age at surgery was 67 years, the average follow up was 29 month. Even if radiological consolidation was incomplete in 18 patients, all patients had a clinically stable and rigid arthrodesis. Mean AOFAS score was 87 (24-100) points at follow up. The FFI was 5.91% (0-66%). Patient satisfaction was excellent in 37 patients (60%), good in 18 (30%), fair in 5(8%) and poor in1 (2%). EQ- 5D was 0.7 (0.4-1).40 patients (66%) estimated their cosmetic result as excellent, 15 (25%) as good, 4(6%) as fair and 2 (3%)as poor. 10 patients (16%) had no shoe wear limitation , 48 (79%) had to wear comfortable shoes and 3 (5%) needed orthopaedic wearing. Professionally 34 patients (56%) had better performances, 18 (26%) had no change and 9 (18%) had aggravation of their capacities but this was due to other health reasons. In sports, 16 patients (26%) had better performances, 35 patients (57%) no change and 10 (17%) were worse as consequence of other health problems for 7. Finally, 56 patients (92%) would recommend the operation and 5 (8%) would not. Conclusion: Experience of clinical practice suggests that the idea of fusing the first MTP joint is initially frequently disregarded by the patients because they fear to be limited by a rigid forefoot. Our results show, in fact, that this procedure can be proposed for numerous pathological situations with the perspective of good to excellent outcome in terms of function and quality of life in the majority of cases.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Transient prenatal expression of NPY-Y1 receptor in trigeminal axons innervating the mystacial vibrissae.

Using immunohistochemistry in combination with confocal laser scanning microscopy, we studied the ontogeny of neuropeptide Y-Y1 receptor (Y1-R) expression in the trigeminal system of the rat. The study was limited to the nerve fibers innervating the mystacial pad and the trigeminal ganglia. In the trigeminal ganglia, Y1-R-immunoreactive (IR) neurons were first observed at E16.5. At this same stage some nerve fibers in the trigeminal ganglia also exhibited Y1-R-like immunoreactivity (LI). Strongly Y1-R-IR nerve fibers innervating the follicles of the mystacial vibrissae were first observed at E18. After double labeling, the Y1-R-LI was found to be colocalized with the neuronal marker protein gene product 9.5. At P1 only weak labeling for the Y1-R was found around the vibrissae follicles, whereas the neurons in the trigeminal ganglia were intensely labeled. The same was true for the adult rat, but at this stage no Y1-R labeling at all was observed in nerve fibers around the vibrissal follicles. These results strongly support an axonal localization of the Y1-R at this developmental stage. The transient expression of the Y1-R during prenatal mystacial pad development suggests a role for the Y1-R in the functional development of the vibrissae.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

Objectives:  Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Methods:  Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Results:  Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. Conclusions:  These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Lateralization of olfactory processing: Differential impact of right and left temporal lobe epilepsies.

Olfactory processes were reported to be lateralized. The purpose of this study was to further explore this phenomenon and investigate the effect of the hemispheric localization of epileptogenic foci on olfactory deficits in patients with temporal lobe epilepsy (TLE). Olfactory functioning was assessed in 61 patients and 60 healthy control (HC) subjects. The patients and HC subjects were asked to rate the intensity, pleasantness, familiarity, and edibility of 12 common odorants and then identify them. Stimulations were delivered monorhinally in the nostril ipsilateral to the epileptogenic focus in TLE and arbitrarily in either the left or the right nostril in the HC subjects. The results demonstrated that regardless of the side of stimulation, patients with TLE had reduced performance in all olfactory tasks compared with the HC subjects. With regard to the side of the epileptogenic focus, patients with left TLE judged odors as less pleasant and had more difficulty with identification than patients with right TLE, underlining a privileged role of the left hemisphere in the emotional and semantic processing of odors. Finally, irrespective of group, a tendency towards a right-nostril advantage for judging odor familiarity was found in agreement with a prominent role of the right hemisphere in odor memory processing.

In vivo visualization of F-actin structures during the development of the moss Physcomitrella patens.

* The 'in planta' visualization of F-actin in all cells and in all developmental stages of a plant is a challenging problem. By using the soybean heat inducible Gmhsp17.3B promoter instead of a constitutive promoter, we have been able to label all cells in various developmental stages of the moss Physcomitrella patens, through a precise temperature tuning of the expression of green fluorescent protein (GFP)-talin. * A short moderate heat treatment was sufficient to induce proper labeling of the actin cytoskeleton and to allow the visualization of time-dependent organization of F-actin structures without impairment of cell viability. * In growing moss cells, dense converging arrays of F-actin structures were present at the growing tips of protonema cell, and at the localization of branching. Protonema and leaf cells contained a network of thick actin cables; during de-differentiation of leaf cells into new protonema filaments, the thick bundled actin network disappeared, and a new highly polarized F-actin network formed. * The controlled expression of GFP-talin through an inducible promoter improves significantly the 'in planta' imaging of actin.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Anti-Nogo-A Antibody Treatment Promotes Recovery of Manual Dexterity after Unilateral Cervical Lesion in Adult Primates--re-examination and Extension of Behavioral Data.

In rodents and nonhuman primates subjected to spinal cord lesion, neutralizing the neurite growth inhibitor Nogo-A has been shown to promote regenerative axonal sprouting and functional recovery. The goal of the present report was to re-examine the data on the recovery of the primate manual dexterity using refined behavioral analyses and further statistical assessments, representing secondary outcome measures from the same manual dexterity test. Thirteen adult monkeys were studied; seven received an anti-Nogo-A antibody whereas a control antibody was infused into the other monkeys. Monkeys were trained to perform the modified Brinkman board task requiring opposition of index finger and thumb to grasp food pellets placed in vertically and horizontally oriented slots. Two parameters were quantified before and following spinal cord injury: (i) the standard 'score' as defined by the number of pellets retrieved within 30 s from the two types of slots; (ii) the newly introduced 'contact time' as defined by the duration of digit contact with the food pellet before successful retrieval. After lesion the hand was severely impaired in all monkeys; this was followed by progressive functional recovery. Remarkably, anti-Nogo-A antibody-treated monkeys recovered faster and significantly better than control antibody-treated monkeys, considering both the score for vertical and horizontal slots (Mann-Whitney test: P = 0.05 and 0.035, respectively) and the contact time (P = 0.008 and 0.005, respectively). Detailed analysis of the lesions excluded the possibility that this conclusion may have been caused by differences in lesion properties between the two groups of monkeys.

Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain.

Changes in expression and function of voltage-gated sodium channels (VGSC) in dorsal root ganglion (DRG) neurons may play a major role in the genesis of peripheral hyperexcitability that occurs in neuropathic pain. We present here the first description of changes induced by spared nerve injury (SNI) to Na(v)1 mRNA levels and tetrodotoxin-sensitive and -resistant (TTX-S/TTX-R) Na(+) currents in injured and adjacent non-injured small DRG neurons. VGSC transcripts were down-regulated in injured neurons except for Na(v)1.3, which increased, while they were either unchanged or increased in non-injured neurons. TTX-R current densities were reduced in injured neurons and the voltage dependence of steady-state inactivation for TTX-R was positively shifted in injured and non-injured neurons. TTX-S current densities were not affected by SNI, while the rate of recovery from inactivation was accelerated in injured neurons. Our results describe altered neuronal electrogenesis following SNI that is likely induced by a complex regulation of VGSCs.