Molecular farming of human tissue transglutaminase in tobacco plants

In this study we have utilized Nicotiana tabacum with a molecular farming purpose in attempt of producing transgenic plants expressing the human tissue transglutaminase (htTG). Three plant expression constructs were used enabling targeting and accumulation of the recombinant protein into the plant cell cytosol (cyto), the chloroplasts (chl) and the apoplastic space (apo). Analysis of transgenic T(0) plants revealed that recombinant htTG was detectable in all three transgenic lines and the accumulation levels were in a range of 18-75 mu g/g of leaf material. In the T(1) generation, the recombinant htTG was still expressed at high level and a significant catalytic activity was detected into the leaf protein extracts. Southern blot analyses revealed that apo and chl plants of T(1) generation possess a high copy number of the recombinant htTG in their genome, while the cyto plants carry a single copy

Structure and in vitro digestibility of grass pea (Lathyrus sativus L.) flour following transglutaminase treatment

The impact of transglutaminase (TG) modification on microstructure and in vitro protein and starch digestibility of grass pea flour was investigated. Results demonstrated that grass pea flour proteins act as effective substrate of TG. Microstructural results showed that the addition of TG produced a more compact structure likely due to TG-catalyzed heteropolymers. Nutritional properties such as slowly digestible starch and expected glycemic index values followed the order: grass pea flour incubated in the absence of TG>grass pea flour incubated in the presence of TG>raw flour. The TG-catalyzed heteropolymers were easily digested as demonstrated by in vitro oral and gastric digestion carried out under physiological conditions. Therefore, TG-modified grass pea flour can be considered as a new source of starch and proteins suitable for feeding a large spectrum of population

Putrescine-polysaccharide conjugate as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine-pectin and -alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine-pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine-polysaccharide derivatives enzymatically crosslinked to proteins were suggested

Trehalose/sodium hyaluronate eye drops in post-cataract ocular surface disorders

Purpose Prospective, randomised, open-label, comparative study to evaluate efficacy of trehalose/sodium hyaluronate eye drops for post-operative discomfort and tear film stability in patients undergoing cataract surgery. Methods Patients with healthy ocular surface, subclinical, or mild dry eye were enrolled. Tear breakup time (TBUT), Schirmer test, dry eye symptoms, corneal fluorescein staining (CFS), and ocular surface disease (OSDI) evaluation were performed pre-operatively and at two and four weeks after surgery. Patients were assigned to receive trehalose/sodium hyaluronate eye drops b.i.d (Group A), or 0.9% unpreserved sodium chloride eye drops b.i.d for 4 weeks (Group B). Results One hundred and thirty-five patients were randomised, 66 patients in Group A (73.2 +/- 4.5 years) and 69 patients in Group B (74.3 +/- 3.8 years), 60.8% females. Fifteen patients (8 Group A) were lost at follow-up. Pre-operatively, no between-group differences were observed, and TBUT increased in Group A between the pre-operative and 2 and 4 week evaluations and was higher in group A than in Group B at 4 weeks. Schirmer test and CFS showed an improvement only in Group A four weeks post-operatively. In Group A an improvement was observed after two and four weeks in foreign body and puncture sensation, whilst a difference in blinking discomfort was observed after four weeks. In Group B we observed an improvement in puncture sensation two and four weeks after surgery. Mean OSDI scores differences between the two groups were significant at four weeks. Conclusions Trehalose/sodium hyaluronate eye drops were effective in reducing signs and symptoms of dry eye and improving tear film stabilit

Advanced platelet-rich fibrin as a therapeutic option in the treatment of dry socket: Literature review and case series

Alveolar osteitis (AO) is one of the complications that occur after tooth extraction. The aim of this study has been to evaluate the efficacy of Advanced Platelet-rich Fibrin (A-PRF) in the management of pain and the acceleration of wound healing in the treatment of AO. Consecutive patients who were diagnosed with AO, recruited from patients referred to the Oral Surgery Department of the University of Naples Federico II, were enrolled. After local anesthesia, the dry socket was curetted and irrigated with saline. The Platelet-rich Fibrin (PRF) clot was placed in the socket and then covered with an A-PRF membrane. Clinical parameters, such as the degree of pain and rate of granulation tissue (GT) formation, were measured before treatment and after 1, 3, 7, 14, and 21 days. The Friedman test for dependent samples was used to detect the treatment and time effect. Four patients with established AO were included. On all the examination days, the post-operative recovery was uneventful. The pain scores progressively reduced, from an average of 8.5 before treatment to 0.25 on the third day, and the GT formation improved over time. The use of A-PRF in the treatment of AO significantly reduced the pain level and enhanced the wound-healing process

Transglutaminase-catalyzed site-specific glycosidation of catalase with aminated dextran

An enzymatic approach, based on a transglutaminase-catalyzed coupling reaction, was investigated to modify bovine liver catalase with an end-group aminated dextran derivative. We demonstrated that catalase activity increased after enzymatic glycosidation and that the conjugate was 3.8-fold more stable to thermal inactivation at 55 ◦C and 2-fold more resistant to proteolytic degradation by trypsin. Moreover, the transglutaminase-mediated modification also improved the pharmacokinetics behavior of catalase, increasing 2.5-fold its plasma half-life time and reducing 3-fold the total clearance after its i.v. administration in rats

Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: Analysis of their mechanical and barrier properties

Chitosan-whey protein edible films with different protein concentrations were prepared in the absence or presence of microbial transglutaminase as cross-linking agent. The films prepared in the presence of the enzyme showed low solubility at a wide range of pH, a lower degree of swelling, and good biodegradability following protease treatments. The presence of transglutaminase induced also an enhancement in film mechanical resistance and a reduction in their deformability. Finally, the barrier efficiency toward oxygen and carbon dioxide was found to be markedly improved in the cross-linked films which showed also a lower permeability to water vapor. Some potential practical applications of transglutaminase-treated chitosan-whey protein films are suggested