Structural and Functional Alterations of Subcutaneous Small Resistance Arteries in Severe Human Obesity

Obese persons are at increased cardiovascular risk and exhibit increased arterial stiffness and impaired endothelial function of large‐ and medium‐size arteries. We hypothesized that normotensive subjects suffering from severe obesity would also present remodeling and endothelial dysfunction of small resistance arteries. A total of 16 lean (age: 49.6 ± 2.9 years, BMI: 22.9 ± 0.3 kg/m², mean ± s.e.m.) and 17 age‐matched severely obese (BMI: 41.1 ± 2.3 kg/m²) normotensive subjects were investigated. None had glucose or lipid metabolic abnormalities except for insulin resistance. Resistance arteries, dissected from abdominal subcutaneous tissue, were assessed on a pressurized myograph. For superimposable blood pressure, the media thickness, media cross‐sectional area (CSA), and media‐to‐lumen ratio values of resistance arteries were markedly and significantly greater in obese compared to lean subjects (media thickness 26.3 ± 0.6 vs. 16.2 ± 0.6 µm, CSA 22,272 ± 1,339 vs. 15,183 ± 1,186 µm², and media‐to‐lumen ratio 0.113 ± 0.006 vs. 0.059 ± 0.001, respectively, P < 0.01). Acetylcholine‐induced relaxation was impaired in vessels from obese subjects compared to the lean individuals (?40.4 ± 1.3%, P < 0.01), whereas endothelium‐independent vasorelaxation was similar in all groups. Stiffness of small arteries as assessed by the stress/strain relationship was similar in lean and severely obese subjects. We conclude that severe human obesity is associated with profound alterations in structural and functional characteristics of small arteries, which may be responsible for the presence of elevated cardiovascular risk and increased incidence of coronary, cerebrovascular and renal events reported in obesity.     

Errors Affect Hypothetical Intertemporal Food Choice in Women

Growing evidence suggests that the ability to control behavior is enhanced in contexts in which errors are more frequent. Here we investigated whether pairing desirable food with errors could decrease impulsive choice during hypothetical temporal decisions about food. To this end, healthy women performed a Stop-signal task in which one food cue predicted high-error rate, and another food cue predicted low-error rate. Afterwards, we measured participants’ intertemporal preferences during decisions between smaller-immediate and larger-delayed amounts of food. We expected reduced sensitivity to smaller-immediate amounts of food associated with high-error rate. Moreover, taking into account that deprivational states affect sensitivity for food, we controlled for participants’ hunger. Results showed that pairing food with high-error likelihood decreased temporal discounting. This effect was modulated by hunger, indicating that, the lower the hunger level, the more participants showed reduced impulsive preference for the food previously associated with a high number of errors as compared with the other food. These findings reveal that errors, which are motivationally salient events that recruit cognitive control and drive avoidance learning against error-prone behavior, are effective in reducing impulsive choice for edible outcomes.     

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.     

Effect of various drugs producing convulsive seizures on rat brain glycerolipid metabolism

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-³H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3–60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciabily vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This lipid effect was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.     

Immunological functions of leptin and adiponectin

Recent years have seen several advances in our understanding of the functions of adipose tissue regarding not only the energy storage, but also the regulation of complex metabolic and endocrine functions. In this context, leptin and adiponectin, the two most abundant adipocyte products, represent one of the best example of adipocytokines involved in the control of energy expenditure, lipid and carbohydrate metabolism as well as in the regulation of immune responses. Leptin and adiponectin secretion is counter-regulated in vivo, in relation to degree of adiposity, since plasma leptin concentrations are significantly elevated in obese subjects in proportion to body mass index while adiponectin secretion decreases in relation to the amount of adipose tissue. In this review we focus on the main biological activities of leptin and adiponectin on the lipid and carbohydrate metabolism and on their contribute in regulation of innate and adaptive immune responses.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from<Emphasis Type="Italic"> Alicyclobacillus acidocaldarius</Emphasis> and<Emphasis Type="Italic"> Archaeoglobus fulgidus</Emphasis> by random mutagenesis and screening

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.     

The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.