Seasonal and intraspecific varation of flavonoids and proanthocyanidins in Cecropia glaziovi sneth. Leaves from native and cultivated specimens

Cecropia glaziovi Sneth. (syn. C. glaziovii, C. glazioui) (Cecropiaceae) is a South American medicinal plant whose antihypertensive activity is attributed to its flavonoid and proanthocyanidin contents. The seasonal and intraspecific variations of these two classes of compounds in C. glaziovi leaves were assayed by spectrophotometry in samples of young and mature leaves collected from native, cultivated and micropropagated trees in the dry and rainy periods of the year. The total flavonoid and proanthocyanidin contents ranged from (0.64 +/- 0.21)% to (3.44 +/- 0.45)% and (2.23 +/- 0.92)% to (5.36 +/- 0.95)%, respectively, among the assayed populations. The flavonoid contents in native plants did not differ statistically between young and mature leaves within the same season, whereas it was higher in both young and mature leaves collected in the dry compared to those collected in the rainy period. For cultivated specimens, the results pointed to higher contents in the dry season, whereas no significant difference was observed for leaves of micropropagated (clone) plants collected in both periods. For the assayed populations, higher proanthocyanidin contents were found in the dry season, excepting the micropropagated (clone) plants, whose contents did not differ significantly between the dry and the rainy periods. Leaves of micropropagated (clone) and cultivated specimens showed less intraspecific variation in the flavonoid and proanthocyanidin contents than those from native trees. These features suggest that, as expected, cultivation of C. glaziovi is of great interest providing raw herbal material of better uniform quality.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Males Benefit from Mating with Outbred Females in Drosophila littoralis: Male Choice for Female Genetic Quality?

The evolution and expression of mate choice behaviour in either sex depends on the sex‐specific combination of mating costs, benefits of choice and constraints on choice. If the benefits of choice are larger for one sex, we would expect that sex to be choosier, assuming that the mating costs and constraints on choice are equal between sexes. Because deliberate inbreeding is a powerful genetic method for experimental manipulation of the quality of study organisms, we tested the effects of both male and female inbreeding on egg and offspring production in Drosophila littoralis. Female inbreeding significantly reduced offspring production (mostly due to lower egg‐to‐adult viability), whereas male inbreeding did not affect offspring production (despite a slight effect of paternal inbreeding on egg‐to‐adult viability). As inbreeding depressed female quality more than male quality, the benefits of mate choice were larger for males than for females. In mate choice experiments, inbreeding did not affect male mating success (measured as a probability to be accepted as a mate in a large group), suggesting that females did not discriminate among inbred and outbred males. In contrast, female mating success was affected by inbreeding, with outbred females having higher mating success than inbred females. This result was not explained by lower activity of inbred females. Our results show that D. littoralis males benefit from mating with outbred females of high genetic quality and suggest adaptive male mate choice for female genetic quality in this species. Thus, patterns of mating success in mate choice trials mirrored the benefits of choice: the sex that benefited more from choice (i.e. males) was more choosy.     

The collagen chaperone HSP47 is a new interactor of APP that affects the levels of extracellular beta-amyloid peptides

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive function that represents one of the most dramatic medical challenges for the aging population. Aβ peptides, generated by processing of the Amyloid Precursor Protein (APP), are thought to play a central role in the pathogenesis of AD. However, the network of physical and functional interactions that may affect their production and deposition is still poorly understood. The use of a bioinformatic approach based on human/mouse conserved coexpression allowed us to identify a group of genes that display an expression profile strongly correlated with APP. Among the most prominent candidates, we investigated whether the collagen chaperone HSP47 could be functionally correlated with APP. We found that HSP47 accumulates in amyloid deposits of two different mouse models and of some AD patients, is capable to physically interact with APP and can be relocalized by APP overexpression. Notably, we found that it is possible to reduce the levels of secreted Aβ peptides by reducing the expression of HSP47 or by interfering with its activity via chemical inhibitors. Our data unveil HSP47 as a new functional interactor of APP and imply it as a potential target for preventing the formation and/or growth amyloid plaques.     

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.     

Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe(182) of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular pi-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.     

Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: preliminary study

The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors.     

Effects of aging and reproduction on protein quality control in soma and gametes of Drosophila melanogaster

In organisms with a soma-germ demarcation, the germline must be 'preserved' such that harmful damage is not transmitted to the offspring. Keeping the progeny free of damage may be achieved by gametes enjoying elevated, and/or more functional, homeostatic maintenance systems. This possibility was approached here by testing whether the soma and maturating oocytes (eggs) dissected from female Drosophila melanogaster in reproductive ages display differential capacities for protein quality control and whether these capacities change during aging and mating. Eggs exhibited a high capacity to prevent protein aggregation, strong capacity for 26S proteasome-dependent degradation and reduced levels of oxidatively damaged (carbonylated) proteins compared to the soma. The capacity to prevent protein aggregation was not affected in either soma or eggs by age and/or mating, while the 26S proteasome capacity declined in the soma but was maintained in the eggs of aged females. However, the levels of carbonylated proteins increased with age in both soma and eggs, and this increase was more pronounced in females allowed to mate continuously. Furthermore, the levels of carbonylated proteins in the eggs of mated flies correlated negatively with the propensity of the eggs to develop into an adult fly. In young flies, mating caused a decrease in 26S proteasome capacity and an increase in protein carbonylation in the soma, but not in the eggs. These results are in line with trade-off theories of aging where aging is considered a consequence of investment in reproduction over somatic maintenance.     

The role of iNOS inhibitors on lung injury induced by gastrointestinal decontamination agents aspiration

Aspiration is a devastating complication during decontamination procedure in poisoning patients. We have investigated whether S-methylisothiourea protects different pulmonary aspiration gastrointestinal decontamination agent-induced lung injury in rats. Forty-two male Sprague-Dawley rats were assigned to one of six groups (n = 7): normal saline, activated charcoal, polyethylene glycol, normal saline + S-methylisothiourea treated activated charcoal + S-methylisothiourea treated and polyethylene glycol + S-methylisothiourea treated. Normal saline, activated aharcoal and polyethylene glycol were instilled into the lungs. The rats received S-methylisothiourea i.p twice daily for 7 days. Serum surfactant protein D, oxidative stress products and inducible nitric oxide synthase expression in the lung were investigated. The aspiration of activated charcoal significantly increased all histopathological scores (P < 0.01). Only peribronchial inflammatory cell infiltration, alveolar edema, and alveolar histiocytes were increased in the polyethylene glycol groups as compared to the normal saline group (P < 0.05). Pulmonary aspiration increased serum malondialdehyde (P < 0.001), and surfactant protein D (P < 0.05) levels and decreased serum superoxide dismutase levels (P < 0.05). S-methylisothiourea treatment decreased all histopathological scores in the activated charcoal treated S-methylisothiourea group (P < 0.01) and only decreased alveolar edema and alveolar histiocytes in the polyethylene glycol-treated S-methylisothiourea group (P < 0.05). S-methylisothiourea treatment reduced elevated oxidative factors, inducible nitric oxide synthase activity and serum surfactant protein D levels. Our findings showed that S-methylisothiourea may be a protective drug against Activated Charcoal and Polyethylene Glycol-induced lung injury.