Anti-glycation effect and the α-amylase,lipase, and α-glycosidase inhibition properties of a polyphenolic fraction derived from citrus wastes

Abstract

The advanced glycation end products (AGEs) constitute a wide variety of substances synthesized from interactions between amino groups of proteins and reducing sugars, which excess induces pathogenesis of chronic diseases. Brazil is the major producer of citrus, a low-cost source of hesperidin, which is a polyphenol recognized for its capacity to inhibit AGEs formation. This is the first work to evaluate the effects of a polyphenolic fraction derived from citrus wastes on the antiglycation and on the inhibition properties of digestive enzymes on the possibility to process these wastes in high value-added products. At concentrations of 10, 15 and 20?mg/mL inhibition of AGEs was higher than 60%. The extracts were able to inhibit by 76% the activity of pancreatic lipase and by 98% the activity of α-glucosidase. For the α-amylase the inhibition capacity was lower than 50%. Strong correlation was obtained among anti-glycation with polyphenolic content and antioxidant capacity.     

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.     

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> Stahl

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18–24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.     

Dose-Finding in the Development of an LPS-Induced Model of Synovitis in Sheep

Models of transient synovitis that can be controlled with antiinflammatory and analgesic drugs have been used to study pain amelioration. To this end, we aimed to determine the dose of intraarticularly administered E. coli LPS that induced signs of synovitis without systemic signs in clinically healthy male castrated sheep (n = 14). In phase 1, a single dose of LPS (0.5, 1.0, 1.5, or 2.0 ng in a total volume of 0.5 mL) was administered into the right stifle joint. In phase 2, a dose of LPS (1.0 or 2.0 μg) in 0.3 mL was administered to 4 naïve sheep. In phase 3, 4 sheep from phase 1 were inoculated after a 60 d washout period with either 0.5 or 1.0 μg of LPS. During the first 48 h after LPS administration, the following were performed: assessment of clinical parameters; scoring for lameness, pain on limb flexion, and local swelling; and ultrasonography of the joints were performed. The doses tested during phase 1 produced subtle signs. During phase 2, mild to moderate lameness with no evidence of systemic signs occurred at both doses. In phase 3, clinical responses were similar between the 0.5- and 1-µg doses. Signs of swelling were not observed at any time. Therefore, we consider the 0.5-µg to be the most appropriate for this model, because it was the lowest dose tested capable of causing lameness without signs of systemic inflammation in all animals.

As an experimental model for the study of arthropathies, the aseptic administration of small doses of endotoxin in the joint induces mild to moderate inflammation and the development of clinical signs similar to those of the naturally occurring disease.⁶ Some studies have used models of transient synovitis to determine whether the associated pain can be controlled with antiinflammatory and analgesic drugs. The use of an LPS-induced model of synovitis to evaluate the analgesic effect of various therapeutic protocols has mainly been reported for horses.^9,16,27,28 However, sheep are an important model species in biomedical research, particularly in orthopedic studies,^15,20,32 due to their similarity in weight, size, and joint and bone structure with humans, and in cardiovascular^7,11 studies, because they are good models of cardiac anatomy and physiology. Consequently, the development of analgesia protocols for acute pain conditions is greatly needed.Animal experiments are under increasing focus regarding their ethical and legal aspects. In vivo studies are permitted when methods consistent with the 3Rs principals (replacement, refinement, and reduction) are considered and implemented.²⁶ This means that experiments have to be performed without animals when possible (replacement) or with as few animals as possible (reduction) and with as little pain and distress as possible (refinement). In this context, species-specific analgesia is considered an important refinement method applicable to the majority of research.²⁵ However, few studies have been conducted to determine analgesia protocols for different pain conditions in sheep. The standardization of animal pain models is necessary for the reliable evaluation of efficient and different drug protocols.^10,19,24To guide standardization of the dose of E. coli LPS for intraarticular administration, with the aim of developing a pain model for studies of analgesia in sheep, we here assessed the ability of various intraarticular doses of LPS to trigger synovitis. Our hypothesis was that the dose established for use in horses (0.5 ng/joint) would trigger similar effects in sheep.