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.     

Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.     

A role for retinal brain-derived neurotrophic factor in ocular dominance plasticity

Visual deprivation is a classical tool to study the plasticity of visual cortical connections. After eyelid closure in young animals (monocular deprivation, MD), visual cortical neurons become dominated by the open eye, a phenomenon known as ocular dominance (OD) plasticity . It is commonly held that the molecular mediators of OD plasticity are cortically derived and that the retina is immune to the effects of MD . Recently, it has been reported that visual deprivation induces neurochemical, structural, and functional changes in the retina , but whether these retinal changes contribute to the effects of MD in the cortex is unknown. Here, we provide evidence that brain-derived neurotrophic factor (BDNF) produced in the retina influences OD plasticity. We found a reduction of BDNF expression in the deprived retina of young rats. We compensated this BDNF imbalance between the two eyes by either injecting exogenous BDNF in the deprived eye or reducing endogenous BDNF expression in the nondeprived eye. Both treatments were effective in counteracting the OD shift induced by MD. Retinal BDNF could also influence OD distribution in normal animals. These results show for the first time that OD plasticity is modulated by BDNF produced in the retina.     

Fractal dimension of the middle meningeal vessels: variation and evolution in Homo erectus, Neanderthals, and modern humans

The middle meningeal vascular network leaves its traces on the endocranial surface because of the tight relationship between neurocranial development and brain growth. Analysing the endocast of fossil specimens, it is therefore possible to describe the morphology of these structures, leading inferences on the cerebral physiology and metabolism in extinct human groups. In this paper, general features of the meningeal vascular traces are described for specimens included in the Homo erectus, Homo neanderthalensis, and Homo sapiens hypodigms. The complexity of the arterial network is quantified by its fractal dimension, calculated through the box-counting method. Modern humans show significant differences from the other two taxa because of the anterior vascular dominance and the larger fractal dimension. Neither the fractal dimension nor the anterior development are merely associated with cranial size increase. Considering the differences between Neanderthals and modern humans, these results may be interpreted in terms of phylogeny, cerebral functions, or cranial structural network.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Study of the surface morphology of a cholesteryl tethering system for lipidic bilayers

The immobilization of functional molecules embedded in lipidic membranes onto inorganic substrates is of great interest for numerous applications in the fields of biosensors and biomaterials. We report on the preparation and the morphological characterization of a tethering system for lipidic bilayers, which is based on cholesteryl derivatives deposited on hydrophilic surfaces by self-assembling and microcontact printing techniques. The investigation of the structural properties of the realized films by atomic, lateral, and surface potential microscopy allowed us to assess the high quality of the realized cholesteryl layers.     

Nitrosylhemoglobin formation after infusion of NO solutions: ESR studies in pigs

A saturated nitric oxide (NO) solution (1.88 mM) infused i.v. in the anesthetized pig at a dose of 68 nmol/kg/min for 24 min resulted in a time-dependent increase of nitrosylhemoglobin [HbFe(II)NO] as determined by electron spin resonance (ESR), reaching a C(max) of 7.99 +/- 0.42 microM at the end of the infusion, compared to 1.13 +/- 0.42 microM before (p < 0.01). This indicates that NO i.v. is efficiently bioconserved as HbFe(II)NO (approximately 34% of the NO dose) and to a greater extent than by the oxidative pathway (approximately 24% of the NO dose), as determined by measuring plasma nitrites/nitrates (chemiluminescence) and Met-Hb (ESR analysis). When the NO infusion was stopped, HbFe(II)NO declined with a t(1/2) of 15 min, indicating that it is a stable storage form of NO, able to deliver NO distally to the site of administration. No significant differences were observed in systemic and pulmonary vascular resistances during and after NO infusion, but PO(2) showed a significant decrease 15 and 30 min after the infusion. Thus, in normoxic/physiological conditions, HbFe(II)NO does not induce significant NO-dependent vasorelaxation.     

Morphological, physiological and behavioural response patterns of carp gudgeon Hypseleotris spp. to food deprivation: implications for assessing health

Morphological (growth, Fulton's condition factor), physiological (per cent dry mass, total lipid content) and behavioural (activity levels) response patterns of carp gudgeon Hypseleotris spp. were examined in response to food deprivation during a 56 day experiment. Considerable variability in the nature and magnitude of these response patterns was observed, suggesting that caution should be taken when interpreting changes in the health of small-bodied fishes based on individual response variables.