ALMS1-deficient fibroblasts over-express extra-cellular matrix components, display cell cycle delay and are resistant to apoptosis

Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions.     

Playing together,laughing together: rapid facial mimicry and social sensitivity in lowland gorillas

In nonhuman animals, the phenomenon of rapid facial mimicry (RFM)—the automatic, involuntary, and rapid (<1 s) replication of others’ facial expressions—has been mainly investigated in the playful domain. In immature lowland gorillas Gorilla gorilla gorilla both play face (PF) and full PF (FPF) are rapidly mimicked between the players. This makes the species suitable to test hypotheses on the factors influencing RFM during play. The observations on 3 captive groups of lowland gorillas (N = 27) revealed that contrary to expectations, the closeness of social bond negatively influenced the occurrence of RFM but it did not affect either RFM latency or its overlapping index (OVERLAP). RFM was affected by the degree of symmetry of play fighting: the more balanced the session, the higher the occurrence of RFM. Players of the same sex class responded faster than players of different sex. These findings suggest that RFM may help synchronizing behaviors of playmates matching in size (same-sex) and promote symmetric playful interactions. “Laughing together” (measured by the RFM OVERLAP) lasted longer when the responder perfectly mirrored the partner expression (PF→PF; FPF→FPF). If PF and FPF convey information on the different play roughness degree, through “laughing together” the players could coordinate their actions and share positive moods and playful intensity. If the perfect congruency in the motor resonance, also known as social sensitivity, can foster a possible emotional dialogue between gorillas remains to be investigated.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

Cell-cycle inhibition by Helicobacter pylori L-asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.     

Luminal adenosine stimulates chloride secretion through A1 receptor in mouse jejunum

Adenosine is known to stimulate chloride secretion by mouse jejunum. Whereas the receptor on the basolateral side is believed to be A2B, the receptor involved in the luminal effect of adenosine has not been identified. We found that jejuna expressed mRNA for all adenosine receptor subtypes. In this study, we investigated the stimulation of chloride secretion by adenosine in jejuna derived from mice lacking the adenosine receptors of A1 (A1R) and A2A (A(2A)R) or control littermates. The jejunal epithelium was mounted in a Ussing chamber, and a new method on the basis of impedance analysis was used to calculate the short-circuit current (I(sc)) values. Chloride secretion was assessed by the I(sc) after inhibition of the sodium-glucose cotransporter by adding phloridzin to the apical bathing solution. The effect of apical adenosine on chloride secretion was lost in jejuna from mice lacking the A1R. There was no difference in the response to basolaterally applied adenosine or to apical forskolin. Furthermore, in jejuna from control mice, the effect of apical adenosine was also abolished in the presence of 8-cyclopentyl-1,3-dipropylxanthine, a specific A1R antagonist. Responses to adenosine were identical in jejuna from control and A(2A)R knockout mice. This study demonstrates that A1R (and not A(2A)R) mediates the enhancement of chloride secretion induced by luminal adenosine in mice jejunum.     

Impurity analysis of retinoic acid samples

The structure of an impurity contained in samples of all trans-retinoic acid was established by means of NMR and MS spectra, and confirmed by X-ray diffraction analysis. The chemical structure of the impurity 2 was found to be strictly correlated to the synthetic procedure employed for the preparation of the retinoic acid samples. Single crystal analysis allowed us to characterise the molecular conformation and the crystal structure of 2.     

Small sense and antisense RNAs derived from a telomeric retroposon family in Giardia intestinalis

Sequencing of a library of small RNAs from Giardia intestinalis identified a novel class of small sense and antisense RNAs homologous to the retroposon family GilT/Genie1 that is located at certain telomeres. These small RNAs may contribute to silencing GilT expression via the RNA interference pathway.