Na(+) channel regulation by calmodulin kinase II in rat cerebellar granule cells

The effects of specific CaM kinase II inhibitors were investigated on Na(+) channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 microM and consisted of an 80% reduction of the peak Na(+) current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 microM), an inactive analog, had no detectable effect. KN-62 was however inactive on Na(+) currents recorded from Chinese hamster ovary cells expressing the type II A alpha subunit. We have also analyzed the inhibitory effects of CaM kinase II 296-311 and CaM kinase II 281-309 peptides. Both peptides (75 microM) induced a maximum peak Na(+) current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284-302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na(+) channel activity in cerebellar granule cells.     

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.     

Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

Upper Limb Evaluation and One-Year Follow Up of Non-Ambulant Patients with Spinal Muscular Atrophy: An Observational Multicenter Trial

Assessment of the upper limb strength in non-ambulant neuromuscular patients remains challenging. Although potential outcome measures have been reported, longitudinal data demonstrating sensitivity to clinical evolution in spinal muscular atrophy patients are critically lacking. Our study recruited 23 non-ambulant patients, 16 patients (males/females = 6/10; median age 15.4 years with a range from 10.7 to 31.1 years) with spinal muscular atrophy type II and 7 patients (males/females = 2/5; median age 19.9 years with a range from 8.3 to 29.9 years) with type III. The Brooke functional score was on median 3 with a range from 2 to 6. The average total vital capacity was 46%, and seven patients required non-invasive ventilation at night. Patients were assessed at baseline, 6 months, and 1 year using the Motor Function Measure and innovative devices MyoGrip, MyoPinch, and MoviPlate, which assess handgrip strength, key pinch strength, and hand/finger extension-flexion function, respectively. The study demonstrated the feasibility and reliability of these measures for all patients, and sensitivity to negative changes after the age of 14 years. The younger patients showed an increase of the distal force in the follow-up period. The distal force measurements and function were correlated to different functional scales. These data represent an important step in the process of validating these devices as potential outcome measures for future clinical trials.

Trial Registration

ClinicalTrials.gov NCT00993161     

Upper Limb Strength and Function Changes during a One-Year Follow-Up in Non-Ambulant Patients with Duchenne Muscular Dystrophy: An Observational Multicenter Trial

IntroductionUpper limb evaluation of patients with Duchenne Muscular Dystrophy is crucially important to evaluations of efficacy of new treatments in non-ambulant patients. In patients who have lost ambulation, there are few validated and informative outcome measures. In addition, longitudinal data demonstrating sensitivity to clinical evolution of outcome measures over short-term periods are lacking.ResultsOur study confirmed preliminary data previously reported regarding feasibility of use and of reliability of the MyoSet and the correlation at baseline between distal strength and clinical outcomes such as FVC, Brooke score, age, and duration since loss of ambulation. A significant correlation was observed between the distal upper limb strength and clinical variables. The sensitive dynamometers (MyoGrip and MyoPinch) and MoviPlate captured a 12-month change in non-ambulant Duchenne muscular dystrophy patients of all ages.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00993161","term_id":"NCT00993161"}}NCT00993161 {"type":"clinical-trial","attrs":{"text":"NCT00993161","term_id":"NCT00993161"}}NCT00993161     

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase'' Npr1'' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar coupling and small angle <Emphasis Type="Italic">X</Emphasis>-ray scattering data

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the γS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.