Nerve growth factor antibodies recognize neurotrophin-3

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.     

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.     

Optimization of culture media to enhance the growth of tissue engineered cartilage

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.     

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer’s disease

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer’s disease.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.     

Facile construction of mutations in Haemophilus ducreyi using lacZ as a counter-selectable marker

Haemophilus ducreyi is a Gram-negative bacterium which is the causative agent of chancroid, an ulcerative sexually transmitted disease. In order to understand the pathogenesis of H. ducreyi disease, studies designed to identify potential virulence determinants and construct mutants deficient in the elaboration of these determinants have been undertaken in several laboratories. At the present time, construction of isogenic mutants is accomplished by electroporation of linearized DNA containing insertionally inactivated H. ducreyi genes followed by selection for the resistance marker encoded on the inactivated gene. In our experience, certain mutants are difficult to construct using this procedure. In the construction of strains containing lacZ as a reporter gene, we observed that the growth of lacZ expressing H. ducreyi was inhibited in the presence of X-gal. We have exploited this observation to develop a new strategy for the construction of isogenic H. ducreyi mutants.     

NT Pro BNP Plasma Level and Atrial Volume Are Linked to the Severity of Liver Cirrhosis

Background and Aims

Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.

Methods

Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.

Results

Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7–1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e'' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006).

Conclusion

NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis.     

Origins and Evolution of the Etruscans’ mtDNA

The Etruscan culture is documented in Etruria, Central Italy, from the 8^th to the 1^st century BC. For more than 2,000 years there has been disagreement on the Etruscans’ biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans’ and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.