Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Early diagnosis of invasive pulmonary aspergillosis in a young immunocompetent patient

A 22-year-old insulin-dependent diabetic male was admitted for diabetic ketoacidosis. He developed hospital-acquired pneumonia (HAP) for which empirical antibiotic and antifungal therapy was started on the ward. On day 6, clinical and laboratory findings worsened, and bronchoalveolar lavage (BAL) was performed. Serum real time-polymerase chain reaction (RT-PCR) indicated invasive pulmonary aspergillosis (IPA) and led to antifungal therapy being initiated 48 hours before the results of the BAL culture were available. Despite early appropriate antifungal therapy, however, the patient died on day 22 while being supported by venovenous extracorporeal membrane oxygenation.     

Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity

During development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of?inhibitory synapses onto pyramidal neurons is?a major component of plasticity in the adult neocortex. In?vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.     

Project DyAdd: Fatty acids in adult dyslexia, ADHD, and their comorbid combination

In project DyAdd, we compared the fatty acid (FA) profiles of serum phospholipids in adults with attention deficit hyperactivity disorder (ADHD) (n=26), dyslexia (n=36), their comorbid combination (n=9), and healthy controls (n=36). FA proportions were analyzed in a 2×2 design with Bonferroni corrected post hoc comparisons. A questionnaire was used to assess dietary fat quality and use of supplements. Results showed that ADHD and dyslexia were not associated with total saturated FAs, monounsaturated FAs, or n-3 polyunsaturated FAs (PUFAs). However, those with ADHD had elevated proportions of total n-6 PUFAs (including γ-linolenic and adrenic acids) as compared to those without ADHD. Dyslexia was related to a higher proportion of monounsaturated nervonic acid and a higher ratio of n-6/n-3 PUFAs. Among females none of the associations were significant. However in males, all the original associations observed in all subjects remained and ADHD was associated with elevated nervonic acid and n-6/n-3 PUFA ratio like dyslexia. Controlling for poorly diagnosed reading difficulties, education, dietary fat quality, or use of FA supplements did not generally remove the originally observed associations.     

Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.     

Nanoelectropulse-induced phosphatidylserine translocation

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis-caspase activation and loss of mitochondrial membrane potential-appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000 Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.     

The herpes simplex virus JMP mutant enters receptor-negative J cells through a novel pathway independent of the known receptors nectin1, HveA, and nectin2

The herpes simplex virus type 1(JMP) [HSV-1(JMP)] mutant was selected for its ability to grow and form plaques in receptor-negative J cells. It enters J cells through a novel gD-dependent pathway, independent of all known HSV receptors, nectin1, nectin2, and HveA. Evidence that the pathway is dependent on a nectin3 binding site on HSV-1(JMP) and requires three mutations in gD rests on the following. We derived monoclonal antibodies to nectin3 and show that J cells express nectin3. HSV-1(JMP) entry and cell-to-cell spread were inhibited by soluble nectin3-Fc, demonstrating that virions carry a binding site for nectin3. The site is either directly involved in HSV-1(JMP) entry, or nectin3 binding to its site affects the gD domains involved in entry (entry site). HSV-1(JMP) entry and cell-to-cell spread in J cells were also inhibited by soluble nectin1-Fc, showing that the nectin1 binding site on gD(JMP) overlaps with the entry site or that nectin1 binding to gD affects the entry site. gD(JMP) carries three mutations, S140N, R340H, and Q344R. The latter two lie in the C tail and are present in the parental HSV-1(MP). HSV-1 strain R5000 carrying the S140N substitution was not infectious in J cells, indicating that this substitution was not sufficient. We constructed two recombinants, one carrying the three substitutions and the other carrying the two C-tail substitutions. Only the first recombinant infected J cells with an efficiency similar to that of HSV-1(JMP), indicating that the three mutations are required for the novel entry pathway. The results highlight plasticity in gD which accounts for changes in receptor usage.