Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).     

Uncoupling of apoptosis and Jun/AP-1 activity in human promonocytic cells treated with DNA-damaging and stress-inducing agents

Earlier studies have indicated that Jun/AP-1 activity is associated with, and probably required for apoptosis induction by DNA-damaging and stress-inducing agents in human myeloid cells. To investigate this possibility, we examined the capacity of continuous treatments with etoposide (10 microM) and camptothecin (0.4 microM), and pulse treatments with X-rays (20 Gy), heat (2 h at 42.5 C) and cadmium chloride (2 h at 200 microM) followed by recovery, to provoke apoptosis and to simulate c-jun and c-fos expression and AP-1 binding in U-937 human promonocytic cells. All these treatments generated apoptosis with similar efficacy (50-60% apoptotic cells at 6 h of treatment or recovery). However, the capacity to increase c-jun and c-fos mRNA levels and to stimulate AP-1 binding was very different, ranging from more than a twelve-fold increase in the case of cadmium, to almost no increase in the case of heat-shock and etoposide. When the cells were pre-conditioned with a soft heat shock (1 h at 42 degrees C) the cadmium-provoked apoptosis was greatly inhibited, but the stimulation of AP-1 binding was not affected. The administration of cAMP-increasing agents also reduced the etoposide- and cadmium-provoked apoptosis. However, cAMP greatly stimulated c-jun and c-fos expression and AP-1 binding when applied together with etoposide (which itself was ineffective), and potentiated the cadmium-induced AP-1 binding. Conversely, retinoic acid abrogated the cadmium-provoked stimulation of AP-1 binding and transactivation capacity, and greatly inhibited the stimulation of binding caused by camptothecin and X-rays. However, retinoic acid did not inhibit the induction of apoptosis by these agents. These results indicate that Jun/AP-1 activity is not necessarily coupled with apoptosis, nor required for apoptosis induction by DNA-damaging and stress-inducing agents in human promonocytic cells.     

Cloning and characterization of the breakpoint regions of a chromosome 11;18 translocation in a patient with hamartoma of the retinal pigment epithelium

Mutations of various tumor suppressor genes, e.g., PTEN, TSC1, and TSC2, are known to be responsible for different inherited diseases presenting with multiple hamartomas, a benign tumor resembling neoplasia that results from faulty organ development. Combined hamartoma of the retinal pigment epithelium (RPE) and retina is a rare, congenital, focal malformation of the fundus. So far, no disease gene has been associated with this disorder. By molecular analysis of an apparently balanced and reciprocal translocation between the short arms of chromosomes 11 and 18, t(11;18)(p13;p11.31), in a patient with hamartoma of the RPE and retina, we selected PAC clones crossing the breakpoints on both derivative chromosomes 11 and 18. For the overlapping chromosome 11 clone, two EST clusters were identified, suggesting the existence of at least two genes in the breakpoint region. We constructed a PAC contig and showed that at least three exons of a novel gene map to the breakpoint region on chromosome 18. Based on the results of FISH analysis with the PAC clones of this contig, we suggest the occurrence of a complex rearrangement.     

High prevalence of pathogenic mutations in patients with early-onset dementia detected by sequence analyses of four different genes

Clinical differential diagnosis of early-onset dementia (EOD) includes familial Alzheimer disease (FAD) and hereditary prion disease. In both disease entities, postmortem brain histopathological examination is essential for unambiguous diagnosis. Mutations in the genes encoding the presenilins (PS1 and PS2) and amyloid precursor protein (APP) are associated with FAD, whereas mutations in the prion protein (PrP) gene are associated with prion disease. To investigate the proportion of EOD attributable to known genes, we prospectively (i.e., antemortem) screened these four genes for mutations by sequencing genomic PCR products from patients with EOD before age 60 years. Family history for dementia was positive (PFH) in 16 patients, negative (NFH) in 17 patients, and unknown (UFH) in 3 patients. In 12 patients, we found five novel mutations (in PS1, F105L; in PS2, T122P and M239I; and in PrP, Q160X and T188K) and five previously reported mutations (in APP, in three patients who were most likely unrelated, V717I; in PS1, A79V and M139V; and in PrP, P102L and T183A) that are all considered to be disease causing. Of these 12 patients, 9 had PFH. This indicates a detection rate of 56% (9/16) in patients with PFH. We found two mutations (APP V717I) in two of the three UFH patients, and only one mutation (PrP T188K) in 1 of the 17 patients with NFH. We conclude that because of the lack of specific antemortem diagnostic markers for FAD and hereditary prion disease, all four genes should be included in a molecular diagnostic program in patients with EOD who had PFH.     

Hyperbaric Oxygen Therapy Can Diminish Fibromyalgia Syndrome – Prospective Clinical Trial

BackgroundFibromyalgia Syndrome (FMS) is a persistent and debilitating disorder estimated to impair the quality of life of 2–4% of the population, with 9:1 female-to-male incidence ratio. FMS is an important representative example of central nervous system sensitization and is associated with abnormal brain activity. Key symptoms include chronic widespread pain, allodynia and diffuse tenderness, along with fatigue and sleep disturbance. The syndrome is still elusive and refractory. The goal of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on symptoms and brain activity in FMS.ConclusionsThe study provides evidence that HBOT can improve the symptoms and life quality of FMS patients. Moreover, it shows that HBOT can induce neuroplasticity and significantly rectify abnormal brain activity in pain related areas of FMS patients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01827683","term_id":"NCT01827683"}}NCT01827683     

Endoscopically visible steam pop during high-energy laser pulmonary vein ablation

A 58-year-old woman with atrial fibrillation underwent laser balloon ablation at our centre. During 12 W ablation in the left superior pulmonary vein, a sudden steam pop was witnessed with displacement of the balloon catheter. Visualisation of the pulmonary vein antrum showed a red discolouration at the last ablation site.The endoscopically assisted laser balloon ablation system (EAS) is a relatively novel technique that is being used to perform pulmonary vein isolation (PVI) in the treatment of atrial fibrillation [1]. The EAS consists of a flexible, compliant balloon for sustained wall contact and a power adjustable laser beam for ablation independent of tissue contact.A 58-year-old woman underwent PVI with the EAS due to drug-refractory, symptomatic and paroxysmal atrial fibrillation. During 12 W ablation at the antrum of the left superior pulmonary vein (LSPV), a sudden steam pop was witnessed, with displacement of the EAS catheter (Fig. 1). Visualisation of the LSPV antrum showed a red discolouration, most likely a haematoma in the antral wall of the LSPV, at the last ablation site. A successful PVI was performed; the red discolouration was still present after 1 h. The patient did not develop symptoms related to the steam pop and echocardiography did not reveal any abnormalities.Open in a separate windowFig. 1Witnessed steam pop during endoscopically assisted ablation. Panel a displays the fifth ablation site in the left superior pulmonary vein (LSPV) where the steam pop occurred. The white ring of exposed tissue is a sign of optimal catheter-wall contact. Panel b displays the LSPV antrum directly after the steam pop. Note the red discolouration which was not present in panel a Steam pops are caused by overheating of myocardial tissue, exceeding 100 ℃, and are preceded by a shift in impedance levels, which cannot be measured with the EAS. Higher energy settings and higher contact force are known to increase the risk of steam pops. Steam pops can lead to tissue disruption and cardiac perforation [2]. However, steam pops appear to be a rare complication with reduced EAS energy settings, which we mostly used in 50 EAS patients, in whom no steam pops were observed [3].